Transforming Growth Factor-Β3 Signaling During Palatogenesis By

Transforming Growth Factor-β3 signaling during palatogenesis

Jamie E. Lane

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in the University of Michigan 2014

Doctoral Committee:

Professor Vesa M. Kaartinen, Chair Assistant Professor Benjamin Allen Professor Yuji Mishina Associate Professor Deneen Wellik

DEDICATION

This thesis is dedicated to my family, friends and mentors along the way.

4/20/2001 5/31/2006 1/11/2008 9/30/2009 12/27/2010

Acknowledgements

The work described in this thesis would not have been possible without the help of many people. I would first like to acknowledge my thesis advisor, Vesa Kaartinen, who has provided me with excellent mentoring and training. Thank you for allowing me to be a part of your laboratory and research, I have learned a tremendous amount over the years. I would also like to thank each of my committee members, Ben Allen, Yuji Mishina and Deneen Wellik for their thoughtful advice and suggestions. The members of the Kaartinen lab have made the laboratory a fun, learning environment, thank you. I would like to personally mention and thank, Penny Thomas for her countless hours of experimental training, advice and suggestions. I would also like to thank Rogerio Castilho and Scott Barolo for helpful discussions on my thesis work. Funding for this work was from the Cell and Molecular Biology Training Grant (NIH-T- 32-GM007315), Rackham Merit Fellowship, Rackham Travel Grants and through NIH grants to Vesa Kaartinen. Tak1 signaling in the craniofacial neural crest-We thank Saverio Bellusci, Rulang Jiang, and YiPing Chen for in situ hybridization probes, Stefan Karlsson for Tgfbr1fx mice, YiPing Chen for sharing unpublished data, Taocong Jin for help with microarray data analyses, Michelle Lynch for Micro-CT analysis, and Sean Edwards and Joseph Helman for support during the study. Tak1, Smad4 and Trim33 signaling in the prefusion palatal epithelium-We thank Jingling Hu for providing IkkaFX mice and Rogerio Castilho for discussions. This work was supported by the NIH R01 grant RO1013085. Tgfb3 expression in the pre-fusion palatal epithelium- We thank Wanda Filipak (University of Michigan Animal Model Core) for preparation of transgenic mice, Scott Barolo for discussions and the NIH-funded FaceBase Constortium and particularly “the genome-wide

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atlas of craniofacial transcriptional enhancers”- project (Axel Visel) for depositing the invaluable data for our disposal. This study was supported by a grant from the National Institute of Dental and Craniofacial Research, National Institutes of Health (DE013085 to Vesa Kaartinen).

Table of Contents

Dedication ...... ii Acknowledgements ...... iii List of figures ...... viii List of tables ...... x Chapter 1: Introduction ...... 1 1-1. Health significance ...... 1 1-2. Early facial development ...... 1 1-3. Palatogenesis ...... 6 1-4. Control of growth and patterning of the secondary palate ...... 8 1-5. Signaling pathways controlling palatal epithelial differentiation and midline seam disappearance ...... 10 1-6. Orofacial clefting ...... 16 1-7. Mouse as a model system to study palatogenesis ...... 21 1-7.1. Evolutionary conservation between mouse and human ...... 21 1-7.2. Mouse embryonic stem cells as a method for genomic manipulation ...... 21 1-7.3. Gene targeting in mouse embryonic stem cells ...... 24 1-7.4. Using gene targeting to create loss-of-function mutation ...... 28 1-7.5. Conditional gene removal ...... 30 1-8. Cre driver lines used to study palatogenesis ...... 35 1-8.1. General use of Cre driver lines ...... 35 1-8.2. Methods of temporal regulation ...... 36 1-8.3. Cre drivers used to recombine floxed genes in the palatal mesenchyme .. 40 1-8.4. Cre drivers used to recombine floxed genes in the palatal epithelium ...... 40 1-9. Summary ...... 43 1-9.1. Smad-4 independent signaling in the cranial facial neural crest ...... 43 1-9.2. Smad-4 independent signaling in the pre-fusion palatal epithelium ...... 43 1-9.3. Tgfb3 expression in the palatal epithelium ...... 44 Chapter 2: TGF-β-activated Kinase I (Tak1) mediates agonist-induced Smad activation and linker region phosphorylation in embryonic craniofacial neural crest-derived cells ...... 45 2-1. Summary ...... 45 2-2. Introduction ...... 46 2-3. Experimental procedures ...... 49 2-3.1. Mice ...... 49 2-3.2. Genotyping ...... 49

2-3.3. Conventional RT-PCR ...... 50 2-3.4. Real-time quantitative PCR ...... 50 2-3.5. Histology, in situ hybridization ...... 50 2-3.6. MicroCT analysis ...... 51 2-3.7. Western blotting ...... 52 2-3.8. Primary craniofacial mesenchymal cell cultures ...... 52 2-3.9. Neural crest stem cell cultures ...... 53 2-3.10. Roller bottle organ cultures ...... 53 2-3.11. Microarray analysis ...... 54 2-4. Results ...... 55 2-4.1. Neural crest-specific Tak1 mutants display mandibular hyperplasia and cleft palate ...... 55 2-4.2. Both canonical and non-canonical TGF-β superfamily signaling pathways are affected in Tak1/ Wnt1-Cre mutants ...... 62 2-4.3. Tak1 mediates C-terminal Smad phosphorylation in craniofacial mesenchymal cells ...... 62 2-4.4. Agonist-induced linker region phosphorylation is affected in palatal mesenchymal cells deficient in Tak1 mutants ...... 67 2-4.5. Tak1 as a TGF-β signal transducer in neural crest-derived facial mesenchymal cells ...... 72 2-5. Discussion ...... 78 2-5.1. Role of Tak1 in craniofacial development ...... 78 2-5.2. Role of Tak1 in TGF-β-induced signaling events ...... 80 2-5.3. Interdependence of TGFβRI and Tak1 signaling? ...... 82 Chapter 3: Tak1, Smad4 and Trim33 cooperatively mediate TGF-β3 signaling during palate development ...... 84 3-1. Summary ...... 84 3-2. Introduction ...... 85 3-3. Experimental Procedures ...... 87 3-3.1. Mice ...... 87 3-3.2. Histology, immunohistochemistry and cell death assays ...... 87 3-3.3. In situ hybridization ...... 90 3-3.4. Real-time quantitative PCR ...... 90 3-3.5. Whole-head roller culture assays ...... 92 3-3.6. Western-blot assays ...... 92 3-3.7. Statistical analysis ...... 93 3-4. Results ...... 93 3-4.1. Epithelium-specific Smad4 mutants display mild defects in palatogenesis93 3-4.2. Simultaneous deletion of TGF-β activated kinase-1 (Tak1) and Smad4 results in anterior and posterior palate defects ...... 94 3-4.3. Additional modifiers of TGF-β signaling during palatal epithelial fusion ...... 97 3-4.4. Expression of a TGF-β signaling target Mmp13 is affected in Tak1:Smad4 and Trim33:Smad4 double conditional mutants ...... 100 3-4.6. Altered gene expression in TGF-β pathway mutants ...... 100

3-4.7. Epithelial cell proliferation and apoptosis are variably affected in Tak1:Smad4 and Trim33:Smad4-dcKOs ...... 103 3-4.8. Inactivation of Tak1 in Trim33:Smad4-dcKOs phenocopies the palate defects seen in Tgfb3-cKOs ...... 106 3-5. Discussion ...... 109 Chapter 4: Control elements targeting Tgfb3 expression to the palatal epithelium are located intergenically and in introns of the upstream Ift43 gene ...... 114 4-1. Summary ...... 114 4-2. Introduction ...... 115 4-3. Experimental Procedures ...... 117 4-3.1. BACs and BAC recombineering ...... 117 4-3.2. Insertion of the SA-lacZ-PA cassette into exon 1 of the 5’ BAC RP23-76M13 and 3’ BAC RP24-299H18 ...... 117 4-3.3. Preparation of the 61-kb and 28-kb BACs ...... 117 4-3.4. Preparation of smaller reporter constructs ...... 119 4-3.5. Alignment of orthologous sequences and identification of putative binding motifs ...... 120 4-3.6. Generation of transgenic mouse lines and transient transgenic mouse embryos ...... 120 4-3.7. Other mouse lines used in this study ...... 120 4-3.8. X-gal staining ...... 121 4-3.9. Histology and immunohistochemistry ...... 121 4-4. Results ...... 123 4-4.1. Palatal peridermal cells are not recombined in a commonly used K14-Cre mouse line ...... 123 4-4.2. Survey of the Tgfb3 cis-regulatory function using recombinant reporter BACs ...... 125 4-4.3. Noncoding evolutionarily conserved sequences within the Tgfb3 gene are not responsible for the MEE-specific gene expression ...... 132 4-4.4. Cis-regulatory elements directing gene expression in the MEE are located in intron 2 of the upstream Ift43 gene ...... 133 4-4.5. An additional cis-regulatory region is located in a 5.3-kb fragment immediately upstream of Tgfb3 exon 1 ...... 138 4-5. Discussion ...... 141 Chapter 5: Conclusions ...... 146 5-1. Summary of findings ...... 146 5-2. Tak1 signaling in the craniofacial neural crest ...... 146 5-3. Tak1, Trim33 and Smad4 signaling in the pre-fusion palatal epithelium ...... 148 5-4. Tgfb3 expression in the pre-fusion palatal epithelium ...... 149 5-5. TGF-β3 signaling during palatogenesis and health significance ...... 150 Bibliography ...... 155

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List of figures

1-1. Cranial neural crest cells migrate to different regions of the developing face ...... 3 1-2. Facial processes in development at E9.5-10.5 ...... 4 1-3. Development of the face in humans ...... 5 1-4. Secondary palate formation ...... 7 1-5. Signaling circuits governing palatal shelf growth and patterning ...... 12 1-6. Molecular control of palatal epithelium ...... 15 1-7. Types of orofacial clefts in humans ...... 17 1-8. Isolated inner cell mass is similar to embryoid bodies in culture ...... 23 1-9. Targeted gene deletion through use of homologous recombination in mouse embryonic stem cells ...... 25 1-10. Generation of mouse germline chimeras from embryoic stem cells ...... 26 1-11. Schematic map for disruption of the Tgfb3 gene ...... 29 1-12. Asymmetric gene targeting ...... 33 1-13. Schematic map for generation of the Tgfb3 floxed allele ...... 34 1-14. R26R-LacZ reporter can be used for lineage tracing ...... 38 1-15. Knock-out, conditional knock-out and inducible conditional knock-out mice ...... 39 2-1. Deletion of Tak1 in neural crest cells leads to mandibular hypoplasia and cleft palate ...... 59 2-2. Palatal shelves of Tak1/Wnt1-Cre mutants elevate and fuse in vitro, but show delayed rugal formation in vivo ...... 61 2-3. Activation of R-Smads and Mapks is less in Tak1/Wnt1-Cre embryos ...... 65 2-4. Tak1 is required for appropriate Smad2 nuclear localization and agonist-induced Smad2/ Smad-3 linker-region phosphorylation in palatal mesenchymal cells ...... 70 2-5. Smad2 linker-region at Thr-220 is phosphorylated by nuclear kinases in palatal mesenchymal cells ...... 74 2-6. Tak1 and TGFβRI mediate both distinct and overlapping gene responses in palatal mesenchymal cells ...... 76 3-1. Epithelium-specific deletion of Tak1 and Smad4 results in defects in palatogenesis ...... 95 3-2. Simultaneous deletion of Trim33 and Smad4 in epithelial cells leads to persistence of the midline seam ...... 98 3-3. Gene expression differences in prefusion and fusing palatal shelves between controls and TGF-β pathway mutants ...... 102 3-4. Proliferation and cell death are affected in Tak1:Smad4 and Trim33:Smad4-dcKOs ...... 104 3-5. Conditional removal of Trim33 and Smad4 along with Tak1 inhibition recapitulates the palatal phenotype seen in Tgfb3-cKO embryos ...... 107 3-6. Schematic representation of redundant functions of Tak1, Smad4 and Trim33 during

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palatal epithelial fusion ...... 108 4-1. Milder palatal phenotype of epithelium-specific Tgfb3:K14-Cre mutants than that of Tgfb3 null mutants results from an inability of K14-Cre to recombine in peridermal cells ...... 124 4-2. Recombinant reporter BACs used to detect Tgfb3 regulatory regions ...... 126 4-3. Enhancer screening across the Tgfb3 region ...... 129 4-4. A 61-kb genomic region including the Tgfb3 gene targets reporter activity to the MEE and adjacent periderm ...... 131 4-5. Evolutionarily conserved regions within the Tgfb3 gene do not direct reporter expression in palatal shelves ...... 135 4-6. Cis-regulatory elements targeting reporter activity to the MEE and adjacent periderm are located in intron 2 of the upstream Ift43 gene ...... 136 4-7. A putative proximal enhancer directing palatal expression lies in a 5.3-kb region upstream of the Tgfb3 gene ...... 139 5-1. LEF1 and FOX sequence alignment to identified cis-regulatory elements of Tgfb3 ...... 151 5-2. Schematic representation of mesenchymal Smad2 activation via Tak1 ...... 152 5-3. Schematic representation of Tak1, Smad4 and Trim33 redundant functions during palatal epithelial fusion ...... 153 5-4. Schematic representation of medial edge epithelium (MEE) specific cis-regulatory elements ...... 154

List of Tables 1-1. Structures contributing to the human face ...... 5 1-2. Selected CL/P syndromes with known genetic cause ...... 20 1-3. Cre-loxP and Flp-FRT systems ...... 32 1-4. Cre driver lines used to study palatogenesis ...... 42 3-1. Crosses used to generate mutant embryos ...... 89 3-2. Real-time quantitative PCR primers ...... 91 3-3. Summary of palatal defects in Tgfb3, Smad4, Tak1 cKOs and Tak1:Smad4 –dcKOs ...... 96 3-4. Summary of palatal defects in Ikka and Trim33 cKOs and Ikka:Smad4 and Trim33:Smad4-dcKOs ...... 99 4-1. Primer sequences used for In-Fusion cloning ...... 122

Chapter 1

Introduction

1-1. Health Significance

Cleft palate is a common human birth defect occurring in about one out of every 2500 babies (Tolarova and Cervenka, 1998). The causes of clefting are varied and are due to combinations of environmental and genetic factors [reviewed by(Dixon et al., 2011)]. In order to learn how to prevent or develop improved treatment strategies, it is necessary to understand the basic development and signaling mechanisms in the formation of the palate (palatogenesis).

1-2. Early Facial Development

The forming mouse face is made up of facial processes (E9.5) that are derived from pharyngeal arches and the frontonasal process, surround the primitive mouth (stomodeum) and form the nose, upper and lower jaw, lip, part of the eye and part of the ear. Pharyngeal arches are composed of the mesoderm-derived core and ectoderm-derived lining, and they are filled with highly migratory ecto-mesenchymal cells derived from the cranial neural crest (CNC).

During the late neurulation stage (~E8.0), CNC cells start to form at the dorsal ridges of the fusing neural tube as a result of instructive signals coming from the neuroectoderm, adjacent ectoderm and underlying mesenchyme. Subsequently, they undergo epithelial to mesenchymal transition, delaminate and migrate ventro-laterally to populate pharyngeal arches and other

developing head structures (beginning at E8.5) (Figure 1-1). Most of the skeletal structures and connective tissue of the head are derived from the CNC, which is quite exceptional, since in the trunk region of the body, the skeletal structures are derived from the mesoderm.

The rapid growth of pharyngeal arches is caused by massive migration and simultaneous proliferation of CNC-derived cells. The growing first pharyngeal arch branches into two sets of processes: paired maxillary and mandibular processes. Bilateral mandibular processes (arches) continue to grow until they fuse in the midline forming the lower jaw (E10). While the maxillary processes continue to grow towards the middle of the developing face, the frontonasal process undergoes morphological changes to form the nose and forehead. The frontonasal process develops two ectodermal thickenings (nasal placodes), which invaginate to form two separate horse-shoe like structures around the nasal pits, which are then subdivided into medial and lateral nasal processes. The maxillary process and medial nasal processes fuse (E10.5) in an anterior- posterior manner and soon after fuse with the lateral nasal process forming two nostrils of the developing nose (E12.5). These two sets of fused processes are joined in the center by fusion of the intermaxillary prominence, which is the tissue from the frontonasal prominence that separates the newly formed nostrils, which results in formation of the primary palate, upper jaw and upper lip (Figure 1-2)((Alappat et al., 2005) and (Ray and Niswander, 2012)). (The processes described above are similar in human, see Table 1-1 and Figure 1-3.) At a slightly later point in development (E13), paired outgrowths of palatal shelves become visible from the maxillary process that ultimately gives rise to the secondary palate.

Figure 1-1. Cranial neural crest cells migrate to different regions of the developing face (Dudas and Kaartinen, 2005).

Lateral and dorsal (left and right) views illustrate the migration of cranial neural crest cells that populate regions of the developing face. The cells delaminate from the dorsal ridges of the forming neural tube and migrate ventro-laterally to populate the developing facial primordia and pharyngeal arches (labeled I-IV and VI). Blue arrows indicate the origin and direction of migration of the cranial neural crest cells that populate different areas of the developing face including the frontonasal process, the maxillary processes and pharyngeal arch 1, which gives rise to the mandibular processes. F, Forebrain; ggl.,ganglion; MA, anterior midbrain; MP, posterior midbrain; opt., optic vesicle; OV, otic vesicle; pom, periotic mesenchyme; R1 to R7, rhombomeres 1 to 7; S1 to S3, somites 1 to 3; SM1 to SM7, somitomeres 1 to 7.

Figure 1-2. Facial Processes in mouse development at E9.5-10.5. (Ray and Niswander, 2012).

(A. lateral view and B. ventral view) At E9.5 the growing first pharyngeal arch gives rise to the paired mandibular (MAND purple) and maxillary processes (MxP green). At this time, the frontonasal prominence (yellow) gives rise to the medial nasal process (MNP) and the lateral nasal process (LNP). c. One day later at E10.5, the MxP and MNP fuse (arrows)(on each side of the fusing processes) and form an invagination and soon after fuse to the LNP forming two nostrils of the developing nose.

Table 1-1 Structures contributing to formation of the human face

Prominence Structures Formed Frontonasal Forehead, bridge of nose, medial and lateral nasal prominences Maxillary (paired) Cheeks, lateral portion of upper lip Medial nasal (paired) Philtrum of upper lip, crest and tip of nose Lateral nasal (paired) Alae of nose Mandibular (paired) Lower lip

Figure 1-3. Development of the face in humans. (Dixon et al., 2011)

Schematic diagrams of the human developing face. a. During the fourth week of development the primitive mouth (purple) is surrounded by the frontonasal prominence (blue), the maxillary (green) and mandibular processes (pink). b. During the fifth week of development, the frontonasal prominence has given rise to the medial and lateral nasal processes that have gone through fusion to give rise to the developing nose. c. By the end of the sixth week of development the medial nasal processes have fused to form the upper lip and the mandibular processes have fused forming the lower jaw.

Palatogenesis

The secondary palate consists of a bony anterior and muscular soft palate in the posterior.

It separates the oral-nasal cavity and allows for efficient feeding, speech and breathing. In humans this process occurs during gestational weeks 8-12 and in mice between embryonic days

E12-E15. The palate is composed of many separate structures coming together to form the complete palate: the primary palate, the secondary palate and the nasal septum (in the anterior).

The anterior secondary palate is an area beginning after the incisor foramen and before the appearance of the eyes when going through the frontal plane. Palatogenesis begins with the growth of the two palatal shelves (of the secondary palate) from the maxillary processes of the first pharyngeal arch (Ferguson, 1988). They are composed mostly of cranial neural crest derived mesenchyme (Ito et al., 2003), layered by a layer of cuboidal epithelium and then covered in flattened cells known as periderm (a transient cell layer that covers all embryonic epithelium prior to the differentiation of the epithelial layer below). The bilateral palatal shelves grow vertically down along the sides of the tongue, then they rapidly elevate to a horizontal position

(within hours in mice) and the two palatal shelves approach each other. Prior to the adherence of the two palatal shelves, the periderm is removed (reviewed in (Dudas et al., 2007)). At this point, the epithelium covering the tips of the palatal shelves that are going to fuse, are known as the medial edge epithelium (MEE). The MEE on each shelf then meet and form the midline epithelial seam (MES). In the anterior nasal side of the secondary palate, the nasal septum fuses with the palatal shelves (Figure 1-4). Once the adherence has taken place, the MES is removed allowing for palatal mesenchymal confluence. Following this time point, the primary palate fuses with the secondary palate.

Figure 1-4. Secondary palate formation. (Lane and Kaartinen, 2014)

Schematic representation of palatal growth and fusion. Palatal shelves (PS) can be first seen as outgrowths of the maxillary processes of the first pharyngeal arch (E11.5). Then they grow vertically down along the sides of the tongue (T; E13.0), rapidly elevate (E14.0), form a contact with each other and with the nasal septum (NS) in the anterior palate (P) (E15.0), and eventually fuse (E16.0).

1-4. Control and patterning of the secondary palate

Many studies have shown that gene expression patterns and gene functions are remarkably different in the developing anterior and posterior palate, which give rise to the hard- and soft palate, respectively (Figure 1-5). The anterior–posterior (AP) boundary in the developing palate is on the level of the most posterior ruga. Rugae are ridges on the hard palate that facilitate the movement of food backwards toward the pharynx.

Anterior palate development: It has been demonstrated that Sonic Hedgehog (Shh), an important morphogen, plays a crucial organizing role in development and growth of the anterior secondary palate.(Lan and Jiang, 2009; Rice et al., 2004) Shh is expressed in periodic stripes on the oral side of the developing palate; these stripes correspond to future palatal rugae (transverse ridges on the hard palate) (Baek et al., 2011; Economou et al., 2012). Abrogation of Shh in the palatal epithelium (by K14-Cre) or its receptor Smoothened (Smo) in the palatal mesenchyme

(using Osr2-Cre) results in cleft palate and reduced expression of a number of genes relevant for palatal shelf growth, e.g., Fgf10, Bmp2, Msx1, and Osr2(Lan and Jiang, 2009). Interestingly, mesenchymal Fgf10 expression is also needed for appropriate epithelial SHH signaling via epithelial Fgfr2b activation, (Rice et al., 2004) thus an Fgf10-Shh signaling axis provides an important regulatory feedback loop between palatal epithelium and mesenchyme to control palatal anterior growth and patterning (Rice et al., 2004). Similarly, Bmp4 in the palatal mesenchyme is required both for Shox2, which is needed for anterior palatal growth

(mesenchymal cell proliferation), (Hilliard et al., 2005; Li et al., 2011) and for Shh expression in the palatal epithelium (Zhang et al., 2002). This provides another signaling loop between the epithelium and mesenchyme to control anterior growth of the secondary palate (Zhang et al.,

2002). Zhou et al. recently demonstrated that deletion of the paired-box gene-9 (Pax9)

transcription factor in the palatal mesenchyme resulted in cleft palate that was caused by defective palatal shelf growth and elevation (Zhou et al.,

2013). In Pax9 mutants, Shh expression in the palatal epithelium, and Bmp4, Fgf10, Msx1, and Osr2 expression in the palatal mesenchyme were dramatically reduced. The authors further showed that a novel knock-in allele, expressing Osr2 in the Pax9 locus (Pax9Osr2KI) was able to rescue the posterior, but not the anterior fusion defects in Pax9mutants. Consistent with the lack of anterior phenotypic restoration, the expression levels of many key genes, e.g., Bmp4, Msx1, and Shh were not rescued. The authors concluded that Pax9 acts upstream or parallel to Osr2 and controls several different pathways including Shh, Fgf10, and Bmp4 during development of the anterior palate.

Oro-nasal patterning of the anterior secondary palate: As outlined above, Shh is specifically expressed in the oral palatal epithelium, while Shh receptors (Ptch and Smo) and downstream effectors (Msx1, Osr1, and Osr2) display a graded expression in the adjacent mesenchyme along the oro-nasal axis(Han et al., 2009; Lan et al., 2004). In contrast, the distal- less homeobox-5 (Dlx5) and Fgf7 genes are specifically expressed in the nasal mesenchyme of the palatal shelf, and the authors suggested that the Dlx5-regulated Fgf7 signaling is critically important in negatively regulating the mesenchymal Shh signaling and palatal oro-nasal patterning(Han et al., 2009) (Figure 1-5).

Posterior palate development: The posterior muscular palate (soft palate) functions during swallowing and speech. In addition to neural crest-derived mesenchymal cells, the posterior palate mesenchyme is populated by myogenic cells derived from the cranial paraxial mesoderm (Zhang et al., 1999). The importance of muscle function to palatogenesis was recently demonstrated by Rot-Nikcevic et al., who showed that mouse embryos lacking striated

muscles display cleft palate (Rot-Nikcevic et al., 2006). Compared to the molecular control of the anterior palate development, much less is known about signaling processes governing the growth and patterning of the posterior secondary palate. The transcription factors Tbx22 and Meox2 are specifically expressed in the posterior palate, while Barx1 and Mn1 are expressed along the entire AP axis, although the expression of these genes is stronger in the posterior than in the anterior mesenchyme,(Bush et al., 2002; Li and

Ding, 2007; Liu et al., 2008) and concordant with the posterior expression domain, mice deficient in Tbx22 suffer from a submucous cleft palate(Pauws et al., 2009). Zhou et al. recently showed that unlike previously thought, Bmp4 is expressed both in the anterior and posterior palatal mesenchyme, and its expression is dependent on Pax9 but not on Msx1 in the posterior palate (Zhou et al., 2013). Moreover, their results implied that both Osr2 expression in the posterior palatal mesenchyme and Shh expression in the posterior palatal epithelium (in the developing sensory papilla) is regulated by Pax9 (Zhou et al., 2013).

1-5. Signaling pathways controlling palatal epithelial differentiation and midline seam disappearance

Along with palatal shelf growth, there is growth of maxillary and mandibular processes.

This allows the tongue to slide down and forward, which is required for palatal shelf elevation and reorientation (Ferguson, 1987). Pioneering studies of Walker and Fraser and their recent detailed refinement and complementation by Yu and Ornitz showed that mechanisms of the anterior and posterior palatal shelf elevation are different (Fraser et al., 1957; Yu and Ornitz,

2011). While the anterior palatal shelves are elevated by a ‘flipping-up’ mechanism, the

posterior palatal shelves undergo tissue-remodeling movement. Molecular mechanisms controlling these events are still poorly known (Bush and Jiang, 2012).

After elevation, the palatal shelves meet in the midline and become adherent (Ferguson,

1988). This event is tightly controlled, because inappropriate adherence prevents palatal shelf elevation resulting in cleft palate (Ingraham et al., 2006; Richardson et al., 2009). Periderm cells, joined to each other by tight junctions, have an important role in controlling palatal shelf adherence and epithelial differentiation (Vaziri Sani et al., 2005);(Yoshida et al., 2012) . This thin (one cell) layer of flattened cells is thought to function as a protective layer or insulator preventing aberrant adhesions. However, the loss of peridermal cells is required at sites of fusion, e.g., the tips of apposing palatal shelves for appropriate epithelial differentiation and adherence (Yoshida et al., 2012). Recent studies have shown that mouse embryos lacking Fgf10, Jagged2, Irf6, Ikka (chuk), or Tbx1 show aberrant oral adhesions between the tongue and palatal shelves,(Funato et al., 2012; Humphreys et al., 2012; Ingraham et al., 2006;

Richardson et al., 2009) and it has been suggested that Jagged2-Notch and p63-Irf6 signaling regulate maintenance of periderm cells during palatogenesis(Casey et al., 2006; Ingraham et al.,

2006; Richardson et al., 2009) (Figure 1-6 (a)).

Figure 1-5. Signaling circuits governing palatal shelf growth and patterning (Lane and Kaartinen, 2014). (a) Epithelial–mesenchymal interactions via Pax9-regulated Shh-Bmp and Shh-Fgf feedback loops control growth and patterning of the anterior palate. (b) Oro-nasal patterning of the anterior secondary palate is regulated via Fgf7-mediated Shh repression. (c) Pax9-regulated expression of Bmp4 and Osr2 in the posterior palatal mesenchyme and Shh in the posterior palatal epithelium is required for appropriate posterior growth of the secondary palate. In addition, Meox2, Barx1, and Mn1-Tbx22 signaling module regulate posterior palatal growth. Gray, palatal mesenchyme; Green, palatal epithelium.

Once the apposing palatal shelves have adhered, the midline epithelial seam (MES) must be removed to obtain a mesenchymal confluence (Ferguson, 1988). This happens primarily via programmed cell death (apoptosis), although epithelial–mesenchymal transformation and/or migration, could also play some role in MES removal (Dudas et al., 2007; Vaziri Sani et al.,

2005; Xu et al., 2006). Nevertheless, it is clear that TGF-β signaling plays a critical role in this process. Tgfb3 is strongly and specifically expressed in the MEE, (Millan et al., 1991; Pelton et al., 1990) and mice deficient in Tgfb3 show 100% penetrant cleft palate (Kaartinen et al., 1995;

Proetzel et al., 1995). Moreover, palatal epithelial cells deficient for genes encoding TGF-β type

I or type II receptors fail to undergo apoptosis resulting in a failure of the MES to disappear

(Dudas et al., 2006; Xu et al., 2006). How Tgfb3 expression is regulated in the MEE is poorly understood. It was recently shown that epithelial-specific Ctnnb1 (the gene encoding β-catenin) mutants displayed cleft palate and loss of Tgfb3 expression suggesting that canonical Wnt signaling via β-catenin is required for appropriate MEE-specific Tgfb3 expression (He et al.,

2011). Venza et al. showed that mutations in the FOXE1 gene result in Bamforth-

Lazarus syndrome characterized with craniofacial defects including cleft palate (Venza et al.,

2011), and that Tgfb3 is a direct target of Foxe1 (Venza et al., 2011).

It is unclear which genetic components are needed for effective downstream signaling of

TGF-β. As outlined above, epithelium-specific deletion of genes encoding TGF-β type I and II receptors resulted in defective palatogenesis (Dudas et al., 2006; Xu et al., 2006). Therefore, epithelium-specific deletion of Smad4, which is a critical intracellular TGF-β signal transducer, was expected to result in a similar phenotype. This was not was observed in these loss-of- function mutants (Xu et al., 2008). Xu et al. further demonstrated that simultaneous deletion of Smad4 and inhibition of p38Mapk resulted in persistent MES in explant cultures suggesting

that both Smad-dependent and Smad-independent TGF-β signaling act redundantly for successful palatal epithelial fusion (Xu et al., 2008). Iwata et al. recently showed that Irf6 is a direct target of Smad4 in the palatal epithelium, and that overexpression of Irf6 in the palatal epithelium rescued the palatal defect seen in epithelial-specific Tgfbr2 mutants (Iwata et al.,

2013). Moreover, they showed that Irf6 is needed to suppress dNp63, which is a prerequisite for p21 (Cip1) expression, cell cycle arrest and subsequent MEE loss (Iwata et al., 2013)

(Figure 1-6 (b)). Whether the same mechanism is true for Tgfb3 mutants is unclear.

Figure 1-6. Molecular control of palatal epithelial differentiation and disappearance (Lane and Kaartinen, 2014). a. Molecular control of palatal epithelial (green) differentiation. Fgf-Notch signaling, P63-Irf6 signaling, Tbx1, and Ikk-α regulate differentiation or the prefusion palatal epithelium (green). b. Medial edge epithelial loss is mediated via a signaling cascade involving TGF-β3, Irf6, p63, and p21in the palatal midline epithelial seam (green).

1-6. Orofacial clefting

Disruptions in any of the signaling pathways that contribute to palatal formation can result in human birth defects. Orofacial fusion defects, such as cleft lip with or without cleft palate

(CL/P) and cleft palate only (CP) are common human birth defects (Figure 1-7). CL/P occurs in

1/1000 human births whereas CP occurs in 1/2500 births (Tolarova and Cervenka, 1998). When

CL/P and CP are present without any other defects or any known maternal environmental exposures, they are known as nonsyndromic (NSCL/P and NSCP) and occur in 70% of cases.

The remaining 30% of cases are syndromic and occur in conjunction with anomalies other than oral clefting. They can be broken down into chromosomal defects, Mendelian syndromes, teratogens and uncategorized syndromes (Schutte and Murray, 1999) (Table 1-2 shows selected syndromes with known genetic cause).

Syndromic orofacial clefting cases can be associated with an inherited genetic trait.

Orofacial cleft phenotypes that are present in multiple family members, particularly in large families are useful for genetic studies. It has been appreciated from the early case history studies that there was a strong genetic component to this abnormality (Schutte and Murray, 1999).

The etiology of cleft lip and cleft palate is often complex. A combination of epidemiological studies, genome-wide association studies and analysis of experimental animal models has started to unravel complex gene-environment interactions that are

Figure 1-7. Types of orofacial clefts in humans (Dixon et al., 2011).

A. Illustrations of types of cleft lip and/or palate. (a.-d.) Unilateral clefts. a. Unilateral cleft of the posterior secondary palate. b. Unilateral cleft of the upper lip. c. Unilateral cleft of the hard palate and the upper lip. d. Unilateral cleft of the hard and soft palate and the upper lip. (e-h.) Bilateral clefts. e. Bilateral cleft of the soft palate. f. Bilateral cleft of the upper lip. g. Bilateral cleft of the hard palate and upper lip. h. Bilateral cleft of the hard and soft palate and upper lip. B. Images of patients with varying degrees of cleft with the description of each image. CL, cleft lip; CP, cleft palate; CPO, cleft palate only.

thought to play critical roles in pathogenesis of many of the cleft lip and cleft palate cases.

Initial studies have identified several environmental components of clefting, including nutritional status and lifestyle of the expecting mother. Studies of gene-environment interactions generally examine large populations and look for an increase in single nucleotide polymorphisms (SNPs) along with the presence of maternal risk factors, such as maternal pre-pregnancy obesity (Block et al., 2013), periconsumptional alcohol consumption (Shaw and Lammer, 1999), maternal cigarette smoking during pregnancy (Lieff et al., 1999), environmental tobacco smoke exposure

(Wu et al., 2014), folic acid fortification (Hashmi et al., 2005) and intake of anti-epileptic drugs

(valproic acid) (Paulson and Paulson, 1981).

Above (Fig. 1-5), I have described molecular mechanisms regulating palatal shelf growth, elevation and fusion (Fig. 1-4). Defects in any of these processes can result in cleft palate.

While some of the disease-causing genes, e.g., IRF6, have been identified in human genetic studies, most of the mechanistic data we currently have are derived from studies examining genetically manipulated mouse models (see section 1-7).

Table 1-2. Selected CL/P syndromes with known genetic cause(Leslie and Marazita, 2013).

1-7. Mouse as a model system to study palatogenesis

1-7.1. Evolutionary conservation between mouse and human

Conservation in genetics measures the sequence similarity between two species. In general, mammalian genomes between different species are remarkably well conserved; for example, the conservation level between the mouse and human is about 40% (Gregory et al.,

2002). This similarity makes it possible to study complex developmental processes, e.g., palatogenesis in the mouse system, when ethical reasons prevent experimentation in humans

(Carver and Stubbs, 1997). The mouse as a mammalian model system is convenient because of their small size, short lifespan (1 mouse year is about 30 human years), short reproduction time

(18 days), large litter size, sequenced genome, and most importantly because of our ability to manipulate the mouse genome. Altogether more than 300 genes have been implicated in palatal fusion either in humans or in experimental animal models (Gritli-Linde, 2007).

1-7.2. Mouse Embryonic Stem Cells as a method for genomic manipulation

The understanding of the potential of embryonic stem cells in transgenic studies and methodological advances in gene targeting have created a framework for genomic manipulation in experimental animal models. Currently, true embryonic stem cells have mostly been established from mouse embryos; thus the mouse has become one of the most widely used animal models in modern biology.

Mouse embryonic stem cell discovery began as early as the 1950s (many good reviews exist describing specific timelines in the progress (Capecchi, 2005; Evans, 2011). Isolation of a pluripotent population of cells was demonstrated through the use of teratocarcinoma cell lines, where a single cell could give rise to a teratocarcinoma containing differentiated tissues (teeth

and hair), suggested that this population of cells could produce an entire embryo. However, the teratocarcinoma cell could not be transmitted through the germline due to its atypical karyotype

(Kleinsmith and Pierce, 1964). The isolated inner cell mass cells from developing mouse blastocysts shared similar characteristics to the teratocarcinoma cells in that they produced differentiated tissues. Yet, they had a normal karyotype and transmitted through the germline

(Evans and Kaufman, 1981)(See figure 1-8). Cell lines established from inner cell mass cells are what are currently known as mouse embryonic stem cell lines (Evans and Kaufman, 1981).

Figure 1-8. Isolated inner cell mass (ICM) is similar to an embryoid body (from embryonal carcinoma) in culture (Evans, 2011). a. The developing blastocyst contains an interior cell mass that lies within the surrounding trophoectoderm. The inner cell mass (ICM) contains primitive extra-embryonic endoderm that is surrounded by Reichert’s membrane, which are parietal extra-embryonic endoderm. When the ICM is isolated from the trophoectoderm, it forms primitive endoderm over its entire surface. b. Embryonal carcinoma (EC) cells in culture form embryoid bodies that contain extra-embryonic endoderm of all types (primitive, visceral and parietal). This shows that EC differentiation is similar to the ICM.

1-7.3. Gene targeting in mouse embryonic stem cells

Gene targeting is a method of incorporating foreign DNA into a specific locus in a cell’s genome via homologous recombination (Thomas and Capecchi, 1987). It was first used by

Smithies et al, to insert test plasmid DNA into the human beta-globin locus (Smithies et al.,

1985), and was subsequently adapted into use in embryonic stem cells by Thomas and Capecchi

(Thomas and Capecchi, 1987).

A strategy to target a specific locus in embryonic stem cells was adopted from earlier studies conducted in yeast (Thomas and Capecchi, 1987). A targeting vector was designed that contained foreign DNA flanked by homology arms (sequence similar to those in a target locus).

The targeting vector also contained positive and negative selection markers critical for enrichment of correctly targeted clones. A positive selection marker (e.g., neor) is (a coding region of a gene) that provides resistance to a cytotoxic antibiotic (e.g., geneticin (G418)), and in a targeting vector it is placed between the homology arms. A negative selection marker (e.g. DT or TK) is a coding region of a gene producing a toxic product and it is placed outside of the homology region. Therefore, a successful recombination results in formation of clones that survive when the positive selection marker is retained and negative selection marker is lost

(Mansour et al., 1988) (Figure 1-9). The embryonic stem cells that contain the correct targeted gene insertion are injected into a pre-implantation mouse blastocyst and transplanted into the uterine horns of a pseudo-pregnant foster-mother. This allows the embryo to develop and be born as a chimeric mouse (Fig. 1-10). This chimeric mouse is bred into the germ-line generating mice that contain a targeted gene modification.

Figure 1-9. Targeted gene deletion through use of homologous recombination in mouse embryonic stem cells. (Nature Education, 2009)

Schematic presentation of gene targeting in mouse embryonic stem cells. A positive (NeoR) and a negative selection marker (TK) gene is added to the target vector sequence. The target vector homologously recombines with the black mouse DNA sequence, which occurs in mouse embryonic stem cells. Only cells (pink) that include the positive and exclude the negative markers survive with the addition of neomycin and ganciclover.

Figure 1-10. Generation of mouse germline chimeras from embryonic stem cells (Capecchi, 2005). a. An embryonic stem (ES) cell line containing the desired mutation is isolated and enriched for using positive and negative selection markers. b. By injecting the correct ES cells into a recipient blastocyst, embryos are formed that potentially contain the desired mutation. These embryos are then placed in a pseudo-pregnant foster-mother who gives birth to the chimeric mice. To aid in the identification of the desired progeny, the ES cells and the blastocyst are derived from mice with different coat color alleles. The chimeric line can then be bred into a line containing the desired mutation.

1-7.4. Using gene targeting to create a loss-of-function mutation

Gene targeting in the mouse is used to alter a specific gene and study its function. The modifications that are generated through gene targeting can be an addition of sequence (knock- in), point mutation of a sequence or deletion (knock-out). Below I will use a Tgfb3 knockout study as an example to show how a mouse mutant lacking the known gene has been use to examine palate fusion and pathogenesis of cleft palate. Tgfb3 is specifically expressed in the midline epithelial seam and when it is removed results in a complete cleft of the secondary palate

(Kaartinen et al., 1995; Proetzel et al., 1995). The sequences surrounding the Tgfb3 gene locus were used to flank the positive selection gene neomycin (neo), which also interrupts exon 6, a crucial exon that is responsible for encoding the mature Tgf-β3 protein (Figure 1-11). The negative selection marker thymidine kinase (tk) was included outside of the region of homologous recombination of a correctly inserted recombinant. The targeting vector is electroporated into embryonic stem cells and positive correctly targeted clones were identified.

The correct recombinant was then used to generate chimeric mice that were bred into the germline generating mice that contain the disrupted exon 6 of Tgfb3.

Figure 1-11. Schematic map for disruption of the Tgfb3 gene (Kaartinen et al., 1995).

The wild-type Tgfb3 locus is shown and exons 6 and 7 and indicated by black boxes. Exon 6 was disrupted by the insertion of a neo cassette from a targeting vector via homologous recombination. The correct targeting was identified through the use of restriction site digests (E, EcoRI; Bs, Bsu36I; H, HindIII; N, NotI; Xh, XhoI; S, SalI) and the use of internal and external probes for Southern detection (grey shaded boxes) .

The resulting Tgfb3 mutant animals have a complete cleft of the secondary palate and therefore are not able to suckle milk, which results in death within a day of birth (Kaartinen et al., 1995; Proetzel et al., 1995). This resulting phenotype displays one disadvantage for studying germ-line knockouts due to the lethality that can result when a gene is completely removed. As it may be advantageous to examine loss of a gene at a later time point or in a specific cell type a method to generate conditional knockout animals was developed (see section 1-7.5.).

1-7.5. Conditional gene removal

Soon after introduction of a homologous recombination based technology to manipulate the mouse genome, Klaus Rajewsky and colleagues introduced yet another powerful methodology, which can be used to introduce both temporally and spatially controlled manipulations in the mouse genome(Rajewsky et al., 1996). This so called Cre-loxP system allows a gene or locus flanked by the loxP sites to be excised in the presence of Cre recombinase. This enzyme from bacteriophage P1 that recognizes so called loxP sequences and catalyzes recombination events (Sauer, 1998). A similar system has been identified in yeast. In this case flippase (Flp) recombinase (like Cre) recognizes a set of Flp Recombinase Targets

(FRT, like loxP) sequences (Sadowski, 1995)(Table 1-3).

LoxP and FRT sites are composed of 34-bp sequences composed of palindromic 5’ and 3’ ends and core sequences are either 6 (FRT) or 8 base pairs (loxP), which dictate their directionality(Grainge et al., 2002). If both pairs of sites are inserted in the same orientation, Cre drives excision of the flanked sequence, leaving behind a single target site. If the target sites flank the sequence in opposite orientations, then an inversion of the flanked sequence will occur

(Figure 1-12).

The conditional removal of a gene is useful to study a gene at a specific time point or within a specific tissue. Similar to the knockout design of Tgfb3, a conditional allele was generated that disrupted exon 6, through the use of a targeting vector that contained both positive

(neo) and negative (Tk) selection markers. LoxP sites were inserted surrounding exon 6 (floxed) and the neo was flanked with FRT sites. Successful gene targeting generates a floxed exon 6

(exon 6 flanked by loxP sites), containing FRT flanked neor (Figure 1-13). The FRT flanked neor sequence that remained in the Tgfb3 floxed animals was removed through mating with a Flp deleter mouse line, that expresses Flp recombinase. The resulting progeny that had both the floxed Tgfb3 and flp recombinase resulted in animals that lacked the neor site and contained only the floxed Tgfb3. The floxed animals were healthy and bred normally.

The Tgfb3 allele can be recombined when a mouse is heterozygous either for the floxed allele or for the germ-line allele and carries a Cre transgene is crossed with a mouse that contains the homozygous floxed Tgfb3 gene.

Table 1-3. Cre-loxP and Flp-FRT. Systems for use in developing conditional gene knockouts are shown. The recombinase drives excision of a gene that is flanked by palindromic sequences that contain a core LoxP or FRT site (italicized and underlined).

Recombinase Target Site

Cre-loxP Cyclization 34 bp loxP sites

recombination ATAACTTCGTATAATGTATGCTATACGAAGTTAT

recombinase

(derived from

bacteria) (Cre)

Flp-FRT Flippase Flp recombinase target (FRT) sites

(derived from TTGATGAAAGAATACGTTATTCTTTCATCAA

yeast) (Flp)

recombinase

Figure 1-12. Asymmetric gene targeting. Diagram showing asymmetric target sites (loxp, for example). Target sites are labeled as red triangles with the point of the triangle showing the orientation of the core sequence. If the orientation is in the same direction and flanking the area to be recombined (yellow rectangle), then it will excise out that region into a separate plasmid, where if the target sites are in opposite orientations, then the target area will be inverted.

Figure 1-13. Schematic map for generation of the Tgfb3 floxed allele (Doetschman et al., 2012). a. The Tgfb3 wild-type locus showing introns 4-7 (white boxes) and the 3’ untranslated region. The black boxes below indicate the probes that were used in Southern blot screening for the correctly targeted mutation. b. The targeting vector is shown with the negative selection gene (Tk), a loxP site, followed by FRT flanked Neo cassette within intron 6 and another loxP site within intron 7. c. The Tgfb3flox allele map is shown along with probes used for Southern analysis (black boxes) and genotyping primers (small black arrows). d. Map showing the Tgfb3 conditional removal upon Cre-mediated recombination. Primers are indicated below (small black arrows) that were used to identify the removal of Exon 6.

1-8. Cre driver lines used to study palatogenesis

1-8.1. General use of Cre driver lines

As discussed above, in Cre-loxP system, Cre drives recombination of a sequence that is flanked by loxP sites allowing a target gene to be removed from a specific tissue. Therefore, the specificity of the system is solely dependent on a Cre driver line, i.e., Cre needs to be expressed strongly and specifically within the genome for it to be useful for driving recombination in a particular cell type at a particular time.

Cre mouse lines can be generated by using traditional transgenic, or gene knock-in techniques. Traditional transgenic mice are easy and fast to produce. Transgenic constructs are relatively small (<20kb) and often contain few control elements, e.g., a tissue-specific promoter and enhancer. However, due to a random integration and lack of locus control elements, they often fail to mimic endogenous expression patterns. BAC transgenics that express Cre are useful because they likely contain most of the cis-regulatory elements and thus can overcome positional effects. BAC transgenic constructs are large (150-300kb) and are not very sensitive for positional effects. However, generation of BAC transgenic constructs and mice is more demanding than that of traditional transgenics. The most reliable method to generate Cre driver lines is to knock in a Cre cassette into a desired locus. However, this is relatively time consuming and expensive when compared to transgenic methods based on random integration.

To test and validate novel Cre transgenic lines, several different reporter mouse lines have been generated (Novak et al., 2000; Soriano, 1999). Perhaps, the most commonly used line was made by inserting loxP-STOP-loxP-reporter cassette into the ubiquitously expressed Rosa26 locus (Soriano, 1999). Once crossed with a Cre driver, the STOP cassette is excised allowing for the Rosa26 locus to drive reporter (e.g., LacZ, YFP etc) expression wherever and whenever Cre

is expressed and active. In order to detect Cre expression (both time and location), a tool that is commonly used in the field is the use of a reporter mouse line. Cre time and location of expression can be detected through the use of a reporter mouse, R26R-LacZ. Targeted gene deletion was used to insert a neo stop cassette flanked by loxP sites, followed by LacZ, just downstream of the Rosa26 locus (Soriano, 1999). The Rosa26 locus was used because it is constitutively active from the preimplantation stage of embryo development and therefore is on in every cell type of the developing embryo. Once crossed with a Cre driver line, the loxP sites are excised and allows for the Rosa26 locus to drive LacZ expression wherever and whenever

Cre is expressed (Fig. 1-14).

1-8.2. Methods of temporal regulation

Cre ERT2 transgenic mice allow inducible activation of floxed alleles in adult mice upon administration of tamoxifen. CreERT2 is a fusion protein between Cre and a mutant estrogen receptor ligand-binding domain that can be induced in-vivo by the small molecule inducer tamoxifen. Tamoxifen is a synthetic steroid that when it binds CreERT2, the protein is translocated to the nucleus where Cre recombines sequences flanked by the loxP sites (Feil et al.,

2009).

Another method that allows spatial and temporal regulation is through the use of the tetracycline regulatory system. In the Tet-on system, the tetracycline-responsive transactivator

(tTA) contains the E. coli tet repressor fused to the transactivation domain of VP16 protein of the

Herpes simplex virus along with a tTA dependent promoter that contains an RNA II polymerase promoter fused to tet operator sequence. tTA dimers bind the tet operator sequence allowing gene transcription. However, in the presence of the inducer doxycycline, tTA undergoes a

conformational change not allowing it to bind the tet operator sequence and thus not allowing gene transcription. In the reverse system, tet-off, there is a sequence change in tTA making it rtTA, where now the gene is only on in the presence of doxycycline. Again, Cre induced recombination only takes place in the presence of loxP sites that are contained in the mouse or the embryos within the mouse that is being administered tetracycline.

Both systems allow for temporal regulation of Cre. (Figure 1-14 shows the difference between a global knock-out, a conditional knock-out and a inducible conditional knock-out).

Driver Cre

ROSA Stop LacZ

Driver Cre

β-galactosidase Blue stain ROSA LacZ

Figure 1-14. R26R-lacZ reporter can be used for lineage tracing.

The ubiquitously expressed Rosa26 locus contains a loxP-STOP-loxP cassette followed by LacZ (loxP sites indicated by black triangles). Once crossed with a Cre driver, the STOP sequence is excised and LacZ is expressed. LacZ expression can be detected by X-gal staining.

Figure 1-15 Knock –out, conditional knock-out and inducible conditional knock-out mice (Friedel et al., 2011). a. When a gene is knocked-out, it is removed everywhere (black). b. When a gene is removed conditionally only specific cells have the gene removed (black). c. Cre can be induced temporally removing the gene a specific time and location (black).

1-8.3. Cre drivers used to recombine floxed genes in the palatal mesenchyme

The most commonly used Cre driver to delete genes in the CNC including the palatal mesenchyme is Wnt1-Cre (Danielian et al., 1998; Rowitch et al., 1998). In Cre-positive embryos recombination begins in pre/early migratory neural crest cells at ~E8.5 and continues through

E11.5 (Danielian et al., 1998; Rowitch et al., 1998). It recombines in all of the cranial neural crest derived cells, which include the craniofacial neural crest and palatal mesenchyme (Chai et al., 2000). Another line, which recombines in post-migratory palatal mesenchymal cells was recently introduced (Lan et al., 2007). This was achieved by knocking in an IRES-Cre-pA cassette into the 3’ untranslated region of the Osr2 gene. Osr2-Cre drives Cre expression at embryonic day 10.5-10.75 within the first pharyngeal arch mesenchyme and later derivatives, which include the palatal mesenchyme (Lan et al., 2007) (Table 1-4).

1-8.4. Cre drivers used to recombine floxed genes in the palatal epithelium

Several different Cre-driver lines have been used to delete floxed genes from the palatal epithelium (Table 1-4). The most widely used driver is K14-Cre, which recombines in ectodermal derivatives including oral/palatal ectoderm beginning at E11 (Andl et al., 2004).

However, this driver is not expressed in the overlying periderm (Chapter 4). Pitx2-Cre driven by the Pitx2 promoter and first pharyngeal arch enhancer fragments (cis-regulatory elements) drives

Cre expression in the palatal and dental epithelium beginning at E11.5. However, its expression is mainly in the posterior of the secondary palate (Xiong et al., 2009). Foxg1-Cre is expressed around E12.5 in the palatal epithelium (a part of the expressing facial and head ectoderm) and other epithelial structures of the head and in the foregut. However, Cre recombination is highly dependent on a genetic background (Hebert and McConnell, 2000); in addition, it is not known,

whether Foxg1-Cre recombines in palatal peridermal cells. Tgfb3-Cre was generated through knock-in of cre into exon1 of Tgfb3. Cre positive embryos show specific recombination in the palatal epithelium including the periderm. However, earlier expression of Tgfb3 in several endodermal, mesodermal and ectodermal derivatives limits its usefulness in studies of palatogenesis (Yang et al., 2008).

Table 1-4. Cre driver lines used to study palatogenesis

Driver Time of Palatal tissue Other tissues Disadvantages for use in recombination examining MES removal

Wnt1-Cre E8.5 Mesenchyme All neural crest Recombines in derived tissues, premigratory/early Neural plate migratory neural crest cells

Osr2-Cre E10-10.75 Mesenchyme Mesonephros, Ectopic activation detected specific in a subset of embryos domains of limb buds, maxillary- mandibular junction

Foxg1-Cre E12.5 Epithelium Dorsal and Recombination pattern ventral highly dependent of genetic telencephalon, background and is poorly developing ear, characterized olfactory epithelium, foregut

K14-Cre E12-14.5 Epithelium All ectoderm- Does not recombine in the derived tissues periderm Tgfb3-Cre E8 Epithelium Heart, octic Early recombination in vesicles, limb several tissues buds, midbrain, somites, tail, lower jaw, tongue Pitx2-Cre E11.5-13.5 Epithelium None Mostly in posterior of secondary palate, early recombination

1-9. Summary

1-9.1. Smad4-independent signaling in the craniofacial neural crest

Tgfb3 is expressed specifically in the palatal epithelium and when it is removed globally it results in a full cleft of the secondary palate. However, when Tgfb3 is removed in the palatal epithelium, it results in a milder palatal phenotype. One hypothesis was that Tgfb3 is expressed within the palatal epithelium and signals to the underlying mesenchyme, but when Tgfb3 was removed in post-migratory neural crest cells (Wnt1-Cre), it did not result in a palatal phenotype

(Lane et al., unpublished and data not shown). Although, the palatal phenotype of Tgfb3 mutants cannot be ascribed as a mesenchymal defect, we were interested in the role of Smad4 independent signaling, since loss of TGF-β receptors (Dudas et al., 2006; Xu et al., 2006) in the craniofacial ectomesenchyme results in severe palatal defects.

TGF-β signaling in craniofacial growth and pattering is well established, yet the details of the signaling mechanisms are not well known. The focus of many TGF-β signaling studies has been on Smad-dependent signaling processes, yet the roles of Smad-independent signaling are just now being addressed. In this thesis (Chapter 2), I address the role of Tak1 in the craniofacial neural crest. I found that Tak1 is required for appropriate activation of p38 Mapk (Smad- independent pathway) and TGF-β/ BMP R-Smads (Smad-dependent pathway) in the neural crest-derived craniofacial ecto-mesenchyme.

1-9.2. Smad4-independent signaling in the pre-fusion palatal epithelium

Tgfb3 is strongly and specifically expressed in the midline epithelial seam and mice lacking Tgfb3 have a complete cleft of the secondary palate due to lack of MES removal.

Epithelial specific deletion of the TGF-β receptors Alk5 and Tgfbr2 led to specific palatal

defects. However, it was surprising that epithelial removal of a critical TGF-β3 transducer,

Smad4, did not result in a palatal phenotype. Xu et al further demonstrated in culture that simultaneous removal of Smad4 and inhibition of p38 led to a persistent midline epithelial seam.

This suggests that both Smad-dependent and Smad-independent TGF-β signaling act redundantly for successful palatal epithelial fusion. In this thesis, I studied the downstream signaling components of the Smad-independent pathway and their role in MES removal (Chapter 3).

1-9.3. Tgfb3 expression in pre-fusion palatal epithelium

Tgfb3 is expressed in the epithelial tips of pre-fusion palatal shelves and is needed for successful palatogenesis. Previously, it has been shown that conditional removal of Alk5 or

Tgfbr2 in the palatal epithelium has a milder palatal phenotype when compared to the complete removal of Tgfb3 (knock-out). I have found in this thesis (Chapter 4) that there is a milder phenotype when Tgfb3 is removed specifically in the palatal epithelium (conditional knock-out) and is likely caused by the inability of the K14-Cre driver to recombine in peridermal cells

(flattened layer of cells that cover the epithelial tips of pre-fusion palatal shelves). It is unknown how Tgfb3 gene expression is specifically directed in the pre-fusion MEE and overlying periderm. In this thesis (Chapter 4), I identified the control elements responsible for palate- specific Tgfb3 expression in the epithelium and in the overlying periderm.

Chapter 2:

TGF-β-activated Kinase I (Tak1) mediates agonist-induced Smad activation and linker

region phosphorylation in embryonic craniofacial neural crest-derived cells

2-1. Summary

Although the importance of TGF-β superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad- dependent (so-called “canonical”) signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-β-activated kinase 1 (Tak1,

Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-β superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-β- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor- mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFβRI

and Tak1 kinases mediate both overlapping and distinct TGF-β2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-β superfamily signaling required for normal craniofacial development.

2-2. Introduction

Members of the transforming growth factor-β (TGF-β) superfamily are involved in many normal and pathologic events including development, inflammation, fibrosis and cancer (Blobe et al., 2000). TGF-βs and bone morphogenetic proteins (BMPs) form two important subgroups within the superfamily(Derynck et al., 1998; Massague, 1998). They bind to largely subfamily- specific membrane-spanning complexes of Type I and Type 2 receptors resulting in activation of components of intracellular Smad-dependent and Smad-independent pathways. In the Smad- dependent pathway, receptor-regulated Smads (R-Smads) become phosphorylated at their C terminals via Type I receptor kinase activity, complex with Smad4, and accumulate in the nucleus where they act as transcriptional co-regulators (Derynck et al., 1998; Massague, 1998).

Initially it was thought that TGF-βs signal exclusively through TGF-β Type I and II receptors

(TGFβRII and TGFβRI) to activate TGF-β R-Smads 2 and 3 and similarly BMPs signal through specific Type I and II receptors to activate BMP R-Smads 1,5 and 8 (Derynck and Zhang, 2003).

However, a recent report showed that at least in certain specific cell lines TGF-βs can also signal via mixed receptor complexes resulting in activation of BMP R-Smads (Daly et al., 2008).

As well as C-terminal phosphorylation at a conserved –SSXS- sequence, more recent research has shown that R-Smads can also be phosphorylated in the linker region between their

Mad homology domains, which is important for both full activation and cessation of activation

(Alarcon et al., 2009; Matsuzaki et al., 2009). Specific residues become phosphorylated as the result of distinct pathways: antagonists, such as EGF, which enable the activated R-Smads to remain in the cytoplasm and be degraded (Kretzschmar et al., 1997); and agonist-induced linker phosphorylation which is required for maximal transcriptional activity of R-Smad/Smad4 complexes as well as for rapid turnover of corresponding R-Smads (Alarcon et al., 2009; Aragon et al., 2011). In this ‘action turnover switch’ model the Thr/Ser residues adjacent to proline-rich sequences in R-Smads (Thr-220 in Smad2) are phosphorylated by nuclear Cdks creating a docking site for transcriptional co-regulators (Pin1 in case of TGF-β R-Smads; Yap in case of

Bmp R-Smads). Moreover, it has been suggested that this phosphorylation may enable subsequent phosphorylation of other linker region residues, such as S250 in Smad2, providing a docking site for ubiquitin ligases, such as, Nedd4L, targeting R-Smads for proteasome-mediated degradation (Aragon et al., 2011; Gao et al., 2009).

In addition to Smad-dependent (canonical) TGF-β/BMP signaling, ligands in the TGF-β superfamily can also activate Smad-independent (non-canonical) pathways resulting in activation of the Map kinase signaling (Derynck and Zhang, 2003). A critical step in initiation of the non- canonical pathway is activation of TGF-β activated kinase-1 (Tak1, Map3k7) by Traf6-mediated polyubiquitination (Sorrentino et al., 2008; Yamashita et al., 2008). Other studies have shown that Tak1 is required for fine-tuning of BMP effects during bone development as well as for

TGF-β-induced NF-κb and JNK activation, and also mediates cytokine-induced Ikkα activation

(Gingery et al., 2008; Hoffmann et al., 2005; Wang et al., 2001).

Although the canonical pathway tends to be treated as the more important, recent studies suggest that the Smad-independent TGF-β signaling plays a more significant role in vivo than

previously thought (Holm et al., 2011; Iwata et al., 2012a). In a mouse model of Marfan syndrome in which TGF-β signaling is elevated, the aortic aneurysm phenotypes are made worse by reducing the canonical pathway component Smad4, and the phenotypes less severe by attenuation of non-canonical pathways (Holm et al., 2011). These results show that not only is the non-canonical pathway of importance in disease mechanisms but that the two pathways somehow interact with one another. Mutations in TGF- β type I and type II receptors (TGFBR1 and TGFBR2) associated with Marfan’s syndrome and Loeys-Dietz syndrome lead to an increase in TGF-β signaling activity in the absence of normal canonical signaling(Cardoso et al., 2012).

The importance of maintaining the correct balance between the pathways for normal developmental events is implied by the presence of craniofacial defects including cleft palate in patients suffering from the Loeys-Dietz syndrome(Loeys et al., 2005) and in a mouse model when Tgfbr2 is genetically removed from the neural crest cell population (which form the principal source of mesenchymal cells in the secondary palate precursor structures, the palatal shelves) resulting in cleft palate in the presence of excessive TGF-β-induced non-canonical signaling (Iwata et al., 2012a).

In the light of the important role played by the Smad-independent pathway implied by these studies, as well as the well-established importance on TGF-β and Bmp signaling in normal craniofacial development in mouse models (Iwata et al., 2012a), we examined the role of Tak1 in craniofacial neural crest development by deleting Tak1 function in pre-migratory neural crest cells using the Wnt1-Cre driver line. The mutant mice display hypoplastic facial structures, and cleft palate, which is caused by a delayed palatal shelf elevation. We found that Tak1 is required for appropriate activation of both p38 Mapk (non-canonical pathway) and TGF-β/Bmp R-Smads

(canonical pathway) in the neural crest-derived craniofacial ecto-mesenchyme. We also show

that Tak1 deficiency results in attenuated TGF-β R-Smad linker region phosphorylation, and that

Tak1 kinase mediates both distinct and overlapping agonist-induced transcriptional responses.

Collectively, these results imply that in neural crest-derived mesenchymal cells Tak1 mediates both canonical and non-canonical arms of the TGF-β superfamily signaling.

2-3. Experimental Procedures

2-3.1. Mice

Tak1-flox mice were generated by flanking a critical exon 2 with asymmetric loxP sites

(Lox66 and Lox71) in opposing orientations (see Fig. 2-1; details will be described elsewhere). Wnt1-Cre (from the Jackson Laboratories) and Tgfbr1FX (kindly provided by S.

Karlsson) mice have been described earlier ((Danielian et al., 1998; Larsson et al.,

2001)).Tak1FX+/Wnt1-Cre+ male mice were crossed with Tak1FXFX female mice to obtain timed pregnancies. The presence of a vaginal plug was designated as embryonic day 0 (E0). DNA for genotyping was prepared from yolk sac or from tail tissue using DirectPCR lysis reagents

(Viagen Biotech). Mouse lines were maintained in mixed genetic backgrounds. All experiments involving the use of animals were approved by the Institutional Animal Use and Care Committee at the University of Michigan at Ann Arbor.

2-3.2. Genotyping

Tak1FXFX mice were genotyped by PCR using the following primer sequences (annealing at 60oC): Tak1FX-sense: 5’-gataccttacactggggacca-3’ and Tak1FX-antisense 5’- ggcattcagttgtggagcatt-3’. Wnt1-Cre and Tgfbr1FX mice were genotyped as previously described

(Danielian et al., 1998; Larsson et al., 2001).

2-3.3. Conventional RT-PCR

To assess the recombination efficiency of the floxed Tak1 locus, the total RNAs were isolated from prefusion palatal shelves harvested at E14.0 or maxillary and mandibular first pharyngeal arch at E11 (RNeasy mini kit; Qiagen), and cDNAs synthesized (Omniscript reverse transcriptase, Qiagen) according to manufacturer's protocols. The following primers were used:

Tak1 exon 1-specific sense primer, 5′-GGGGATCATGTCGACAGCCTC-3′; Tak1 exon 3- specific antisense primer, 5′-GTTCACACGTGACAACTGCCG-3′; and Tak1 exon 4-specific antisense primer, 5′-GCATGCTGTGCAGGTAAGCCA-3′. β-Actin was used as a quality and loading control: sense primer, 5′-GTGGGCCGGTCTAGGCACCAA-3′; and antisense primer,

5′CGGTTGCCTTAGGGTTCA-GG-3′.

2-3.4. Real-time quantitative PCR

Total RNAs were isolated, and cDNAs were synthesized as outlined above. Real time quantitative PCR experiments were carried out using Universal Probe Library-based assays

(Roche Applied Science) with gene-specific primer sequences generated by the manufacturer's online algorithm and TaqMan Universal PCR master mix (Applied Biosystems). 30-µl assays were quantified using the ABI7300 PCR and detection system (Applied Biosystems) and analyzed using 7500 System v1.2.2 software.

2-3.5. Histology, in Situ Hybridization, Cell Death, and Proliferation Assays

For histological analyses, tissues were processes, sectioned and stained by H&E according to standard protocols. For whole-mount in situ hybridization, the tissues were fixed in

4% buffered formaldehyde for 12-16 hours and dehydrated though a graded methanol series

(20%, 50%, 70%, 95% and 100%) containing PBST. Antisense RNA probes were synthesized with NTP DIG RNA labeling mix (Roche Applied Science) following manufacturer’s instructions. Probe templates for Shox2, Tbx22 and Shh were obtained from Y-P Chen(Yu et al.,

2005), R. Jiang (Liu et al., 2008) and S. Bellusci (Bellusci et al., 1997), respectively. A probe template for Tgfb3 was prepared as described (Dudas et al., 2004). Apoptotic cells were detected using TUNEL assay (Dead End from Promega) following manufacturer’s instructions. For cell proliferation analyses, cell proliferation labeling reagent (RPN201, Amersham) was used. BrdU- positive cells were detected using anti-BrdU antibody (RPN202, Amersham). Fluorescent images were viewed on an Olympus BX51 microscope and documented using an Olympus DP71 camera.

2-3.6. MicroCT analyses

Specimens were embedded in 1% agarose, placed in a 19-mm-diameter tube, and scanned over the entire length of the skull using a microCT system (µCT100; Scanco Medical,

Bassersdorf, Switzerland). The scan settings were: voxel size 10 µm, medium resolution, 55 kVp, 109 µA, 0.5 mm AL filter, and integration time 500 ms. Images were created using the manufacturer's evaluation software and a fixed global threshold to segment bone from non-bone.

Skull shape was analyzed by measuring the distance from the supraoccipital bone to the anterior end of the frontal bone (length) and from the top of the parietal bone to the cranial base (height) and calculating the height/length ratio. Mandibular length was measured as shown in Fig. 2-1.

2-3.7. Western Blotting

Tissues or cells were lysed in 2xLaemmli sample buffer (Harlow and Lane, 1988), quantified by Quant-It protein assay system (Invitrogen), and samples (5 µg of protein per lane), run on NuPage 4-12% Bis-Tris gradient gels (Invitrogen) and transferred by “iBlot dry blotting”

(Invitrogen) onto nitrocellulose filters. Immonoblotting and detection were done according to standard protocols. Documentation and quantification was accomplished by using the UVP

BioSpectrum AC imaging system. Antibodies used were: pTak1 (#9339 – Cell Signaling),

Smad2 (#5339 –Cell Signaling), pSmad2-C (#3101 –Cell Signaling), Smad1 (#6944 –Cell

Signaling), pSmad1/5/8-C (#9511 – Cell Signaling), p-p38 Mapk (#4511-Cell Signaling), p-Jnk

(#4668- Cell Signaling), p42/44 Erk (#4376-Cell Signaling), β-Actin (#A1978 – Sigma-Aldrich), pSmad2-L(S250) (#35741 – Immuno-Biological Laboratories), pSmad2-L(T220) and pSmad3-

L(T179) (#28087 – Immuno-Biological Laboratories), Histone H3 (#4499-Cell Signaling),

PSMA2 (#2455-Cell Signaling).

2-3.8. Primary craniofacial mesenchymal cell cultures

Mandibular and maxillary processes of the first mandibular arch, and prefusion palatal shelves were dissected from E11.0 and E14.0 embryos, respectively. The cultures were established as described by Thomas et al. (Thomas et al., 2010). The neural crest origin and purity of cultures was confirmed by establishing cultures of embryos that carried both the Wnt1-

Cre transgene and were positive for the R26-lacZ reporter (Fig. 2-3B). β-galactosidase staining was carried out as described (Behringer, 2003). After 7-10 days cells were stimulated with growth factors (TGF-β2, 10ng/ml or BMP2, 100ng/ml) for 0, 10 or 40 minutes, and harvested for

western blot analyses. Chemical inhibitors Flavopiridol (Cdk inhibitor; 1 or 10 µM; Sigma-

Aldrich), UO126 (Erk1/2 (and Jnk) inhibitor; 10mM; Sigma Aldrich), SB431542 (TGFβRI inhibitor; 10 µM; Sigma-Aldrich), 5Z-7-Oxozeaenol (Tak1 inhibitor; 2 µM; eMolecules) and

SB202190 (p38 Mapk inhibitor; 10 µM; Sigma-Aldrich) were added to cells 60 min prior to

TGF-β2 or BMP2 addition. Cells were not starved of serum prior to these treatments. Some cultures were transduced with replication-deficient recombinant adenoviral preparations using

MOI 1000. Ad-Gfp and Ad-Cre (titer, 4 x 1012 vp/ml) were obtained from the University of

Michigan Biomedical Research Core Facility. For immunofluorescence, cultured cells were fixed in 4% buffered formaldehyde (5 mins) and stained using anti-Smad2 (#5339, Cell signaling) antibody. Binding was detected using Alexafluor-594 goat anti-rabbit secondary antibody

(Invitrogen), and slides mounted in Vectashield with DAPI (Vector Labs Inc.). Fluorescent images were viewed and documented as outlined above. In some experiments, nuclear and cytoplasmic fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction kit (#

78833-Thermo Scientific) according to the manufacturer’s instructions.

2-3.9. Neural crest stem cell cultures

The neural crest stem cell line O9-1 was obtained from M. Ishii. Undifferentiated cells were cultured in the presence of LIF and bFGF in SNL-cell-conditioned medium as described

(Ishii et al., 2012).

2-3.10. Roller bottle organ cultures

Heads from embryos were collected at E13.5 and mandible, tongue and brain removed in

PBS. The resulting mid-face samples were cultured for 24-48 hours at 37oC in roller bottles (60

rotations per minute) in serum-free BGJb medium without Pen-Strep. The bottles were gassed at the beginning of the culture and every 12 hours by gently bubbling the medium for 2 minutes with O2/CO2 (95%/5%). The palatal cultures were fixed, sectioned, and stained as described above.

2-3.11. Microarray analyses

Palatal mesenchymal cells were isolated and aliquoted to 4 groups as shown in Table 2-1

Confluent cultures were incubated in the presence of inhibitors (TGFβRI inhibitor SB431542, 10

µM; Tak1 inhibitor 5Z-7-oxozeaenol, 2 µM) for 1 h and then stimulated with TGF-β2 (10 ng/ml) for 2 h. Total RNAs were then isolated using an RNeasy kit (Qiagen). Concentration and purity were determined by spectrophotometry, and RNA integrity was confirmed by running aliquots on 1% agarose gel. 500 ng of total RNA of each sample was amplified and biotin-labeled according to the GeneChip 3′ IVT expression kit user manual (Affymetrix). Microarray analysis was performed with the GeneChip mouse genome 430 2.0 array (Affymetrix). The biotin-labeled

RNAs were fragmented, and 10 µg of each was hybridized to a GeneChip at 45 °C for 16 h.

Labeled bacterial RNAs of known concentrations were spiked in hybridization to generate an internal standard and to allow normalization between the chips. The chips were washed and stained with streptavidin R-phycoerythrin (Molecular Probes). After scanning the chips by

GeneChip Scanner 3000 7G System, the data were analyzed by Affymetrix GeneChip-related software packages GCOS, Data Mining Tool, and Affymetrix web database “NetAffx.”

Microarray data were submitted to the GEO repository under accession number GSE45491.

2-4. Results

2-4.1. Neural crest-specific Tak1 mutants display mandibular hyperplasia and cleft palate

A recent study demonstrated that haploinsufficiency of Tak1 in neural crest cells rescued the cleft palate phenotype of Tgfbr2 mutant mice(Iwata et al., 2012a). However, the role of Tak1 itself in craniofacial development is currently unclear. To address this question, we crossed mice homozygous for the floxed Tak1 allele (Tak1FXFX; for details see Fig. 2-1A) with transgenic

Wnt1-Cre mice that also were heterozygous for the floxed Tak1 allele (Tak1FX+/Wnt1-Cre+). The expected Mendelian proportion of Tak1FXFX/Wnt1-Cre+ mice (hereafter referred to as Tak1/Wnt1-

Cre mutants) survived to birth (n=16/66) but then died within 24 hours.

We used micro-CT analysis to compare the structure of control and Tak1/Wnt1-Cre mutant littermate heads at P0 in detail. The craniofacial skeleton appeared generally stunted in mutants (Fig. 2-1C). Many of craniofacial bones are largely derived from neural crest cells, including cranial base, mandible, maxilla, and frontal skull (Ishii et al., 2012). Although the average lengths of control and Tak1/Wnt1-Cre mutant cranial bases did not differ, those of the mandible and maxilla were about 20% and 5% shorter, respectively, in the mutants (Fig. 2-1C).

The skulls in mutants were also shorter than in controls (length vs height ratio 1.74+/-0.04

(average+/-sem) and 1.49+/-0.02 in controls and mutants, respectively; n=3). The mutants also displayed cleft secondary palate with high penetrance (13/16, 80%).

To investigate the mechanism underlying the palatal defect, we collected mutant and control embryos at defined time points and analyzed palatal phenotype in detail by histology (Fig. 2-1D-

F). At E13.5, the palatal shelves in both control and mutants were still growing vertically towards the floor of the mouth, parallel to the sides of, and separated by, the tongue, which was

almost square in cross section (Fig. 2-1D). By E14.5 (Fig. 2-1E), in controls the tongue had changed shape in cross section, becoming wider than it was tall. It was no longer interposed between the palatal shelves which had elevated to point towards one another parallel to the top of the tongue, and commenced fusion with one another along the midline of the oral cavity in the mid- and posterior regions, though a ‘seam’ of adhered epithelia from each shelf still remained.

In contrast, in Tak1/Wnt1-Cre mutants, the tongue remained interposed between the palatal shelves, lying close to the nasal septum, and was still relatively square in cross section. The shelves had failed to elevate very much. By E18 (Fig. 2-1F), in controls the secondary palates had fused completely along their entire length: the seam formed where their epithelia had met in the midline had disappeared so their mesenchymal cores were confluent, and differentiation into cartilage commenced. In most Tak1/Wnt1-Cre mutants examined, secondary palate formation had failed, leaving a cleft from anterior to posterior. Although the tongue was no longer completely interposed between the palatal shelves, which had elevated, the shelves had failed to adhere to or fuse with one another, although some cartilage formation had occurred. Although now above the tongue in anterior and mid positions, the underside of the shelves and the top of the tongue were very close compared to the same structures in controls.

As components of the head were smaller in mutants than controls at birth, we examined the relative size of palatal shelves in littermates at E13.5, and found that shelves in mutants were about 20% smaller in cross-sectional area than those in controls (Fig. 2-1G, left). BrdU incorporation assays on pre-fusion palatal shelf sections from littermates showed a lower average value in mutant versus control cells at the anterior level (not significantly different), but no difference at the mid and posterior levels (Fig. 2-1G right). To determine if the smaller mutant shelf area at E13.5 was the result of an earlier lower cell proliferation rate, we performed the

same assay at E11 on mandibular primordia (first pharyngeal arch mesenchyme) but no significant difference between control and mutant tissues was detected (data not shown). No differences in cell death were detected at E11 or E13.5 (data not shown)

Since Tak1/Wnt1-Cre mutants displayed a distinct reduction in craniofacial size at birth

(Fig. 2-1) likely resulting in a hypoplastic oral cavity, and the position and shape of the tongue appeared abnormal in mutants during palatogenesis, we wondered whether the failure in palatal shelf elevation could be caused by the failure of the tongue to ‘descend’ appropriately. To address this we cultured mouse embryonic heads at E13.5 (when palatal shelves are still growing vertically), and from which we had removed the mandible and tongue. This was performed in roller bottles under chemically defined conditions. In both control and mutant samples sufficient elevation of palatal shelves occurred after 24 hours that, after a further 24 hours culture, fusion occurred, with equivalent mesenchymal confluence regardless of genotype (Fig. 2-2A-B). This suggests that in Tak1/Wnt1-Cre mutants the tongue obstructed palatal shelf elevation in vivo, contributing to the pathogenesis of cleft palate in Tak1/Wnt1-Cre mutants in vivo (see the manuscript of Song et al., co-submitted with this manuscript).

Previous studies have shown that TGF-β superfamily signaling is involved in the anterior-posterior patterning of the secondary palate (Hilliard et al., 2005; Liu et al., 2005; Yu et al., 2005; Zhang et al., 2002). Therefore, we compared expression patterns of established anterior

(Shox2) and posterior (Tbx22) mesenchymal markers between Tak1/Wnt1-Cre mutant and control littermates using whole mount in situ hybridization. These experiments did not demonstrate detectable differences in Shox2 and Tbx22 expression patterns or intensities between controls and mutants (Fig. 2-2C) suggesting that in Tak1/Wnt1-Cre mutants the anterior-posterior patterning is not grossly affected.

Mesenchymal Bmp signaling has been shown to be important for maintenance of Shh expression in the rugae of the palatal epithelium (Zhang et al., 2002), which in turn plays a critical role in regulation of growth and patterning along the palatal oro-nasal axis (Han et al.,

2009; Rice et al., 2004). Our experiments demonstrate that, unlike palatal mesenchyme-specific

Bmpr1a mutant shelves (Baek et al., 2011), there was no reduction in Shh expression in the primary palate of Tak1/Wnt1-Cre mutants. However, while the amount of Shh expression in individual ruga was not decreased, at the time of palatal fusion (E14.5-E15.5) a marked delay in rugal formation suggests that the rate of expansion of the secondary palate was reduced (Fig. 2-

2D).

In summary, Tak1 deficiency in craniofacial neural crest resulted in facial (particularly mandibular) and palatal shelf hypoplasia, and cleft palate. Palatal shelf elevation was delayed, and the tongue abnormal in position and shape in mutants immediately prior to the normal stage of palatal fusion. No difference between control and Tak1/Wnt1-Cre mutants in anterior- posterior patterning of pre-fusion palatal shelves was detected by marker gene analysis, but a delay in ruga pattern formation suggests abnormal morphogenesis or maturation there. In rolling culture, removal of the tongue and mandible resulted in mutant palatal shelf elevation and fusion occurred in both control and mutant heads.

Figure 2-1. Deletion of Tak1 in neural crest cells leads to mandibular hypoplasia and cleft palate. A, schematic presentation of the gene-targeting strategy. “Flipping” of exon 2, which encodes the kinase domain of Tak1, causes alternative splicing between exons 1 and 3 (B) inactivating the function of Tak1. B, RT-PCR analysis of RNA samples harvested from prefusion (at E14) palatal shelves of control (Tak1FX/+/Wnt1-Cre−) and mutant (Tak1FX/FX/Wnt1-Cre+) embryos. Left panel, exon 1-specific sense and exon 3- specific antisense primers result in amplification of 261-bp wild-type and 150-bp mutated PCR products, respectively. Exon 1-specific sense and exon 4-specific antisense primers result in amplification of 448-bp wild-type and 337-bp mutated PCR products, respectively. C, lateral and superior μCT images of control (Tak1FX/+/Wnt1-Cre−) and mutant (Tak1FX/FX/Wnt1-Cre+) heads at P0. The yellow arrows in the lateral images depict the length of the mandible. The yellow arrows in the superior views depict the length of the maxilla. The red arrows in the superior images depict the length of the cranial base, and the green arrows illustrate palatine bones in a control (fused palate) and mutant (cleft palate). The bar graph shows lengths of the cranial base, maxilla, and mandible of controls (blue columns) and mutants (red columns) (n = 3). D–F, histological comparison of palatal phenotypes of controls and Tak1/Wnt1-Cre mutants: frontal sections at anterior, middle, and posterior levels at E14.5 (E), E18.0 (F), and the level of the first molar at E13.5 (D). between controls and mutants. Middle panel, bar graph shows relative quantification of palatal shelf (PS) size in controls (C, blue columns) and mutants (M, red columns) (n = 3).

The black arrows point to tips of palatal shelves, the black arrowheads point to the midline seam (in a control), and the black vertical lines depict the distance between the tongue and nasal septum. G, left-hand panel shows sections from two independent control and mutant samples at the level of the first mandibular molar. The green highlighting illustrates the area measured to compare palatal shelf size between controls and mutants. Middle panel, bar graph shows relative quantification of palatal shelf (PS) size in controls (C, blue columns) and mutants (M, red columns) (n = 3). Right panel, bar graph summarizes results from BrdU incorporation assays on controls (blue columns) and Tak1/Wnt1-Cre mutants (red columns) palatal shelves at E13.5 (n = 4). Error bars, S.E. *, p < 0.05. NS, nasal septum; P, palatal shelf; SP, secondary palate.

Figure 2-2. Palatal shelves of Tak1/Wnt1-Cre mutants elevate and fuse in vitro, but show delayed rugal formation in vivo. A and B, control (left panels) and mutant (right panels) embryos were harvested at E13.5, and dissected midfacial segments (without mandible, tongue, and brain) were cultured under chemically defined conditions in roller bottles for 24 h (A) or 48 h (B). The arrows point to the tips of elevated palatal shelves in both control and mutant samples cultured for 24 h (n = 3). C and D, comparison of Shox2 and Tbx22 (C, E14.0) and Shh (D, E14.5 and E15.5) expression in control and Tak1/Wnt1-Cre mutant palatal regions. The double-headed arrows in C illustrate the length of the positive signal. Rugal stripes (positive Shh expression) were identified and numbered (r1–r8) according to(Economou et al., 2012). E–H, n = 3. NS, nasal septum; P, palatal shelves; SP, secondary palate.

2-4.2. Both Canonical and Non-canonical TGF-β Superfamily Signaling Pathways Are Affected in Tak1/Wnt1-Cre Mutants

Neural crest cells make a contribution to all the craniofacial structures identified as showing abnormalities in Tak1/Wnt1-Cre mutants, including palatal shelves, tongue and mandible. Of these, the palatal shelf mesenchyme is the most uniformly composed of neural crest cells. To identify signaling processes influenced by Tak1 deficiency in vivo, we harvested prefusion palatal shelves from control and Tak1/Wnt1-Cre embryos and analyzed their protein for Tak1, phosphorylation of Tak1 and its downstream signaling target p38 Mapk, the TGF-β signaling target Smad2 and BMP signaling targets Smad1/5/8 (Fig. 2-3A). The level of Tak1 and

Tak1 C-terminal phosphorylation at Ser-412 (S412) was significantly less in Tak1/Wnt1-Cre mutant samples demonstrating an efficient Cre-induced recombination in the Tak1 locus. The level of p38 Mapk phosphorylation was also markedly and consistently less in these mutant palatal shelves when compared to corresponding control samples. Similarly, C-terminal Smad2 phosphorylation was consistently lower in mutant shelves, while there were no differences in

Smad2 protein levels. C-terminal Smad1/5/8 phosphorylation was lower on average in mutant samples but not significantly so. These results show that Tak1 deficiency leads to decreased signaling activity in both non-canonical and canonical TGF-β signaling pathways.

2-4.3. Tak1 Mediates C-terminal Smad Phosphorylation in Craniofacial Mesenchymal Cells

To gain a deeper insight into processes regulated by Tak1-mediated signaling in postmigratory neural crest cells, we established control andTak1/Wnt1-Cre mutant craniofacial mesenchymal primary cultures from both prefusion palatal shelves (at E14) and first pharyngeal arches, which give rise to maxilla and mandible (at E11), and analyzed their ability to respond to

TGF-β stimulation (Fig. 2-3, B and C). As expected, control mesenchymal cells isolated from the secondary palate showed an increase in Tak1 Ser-412 phosphorylation in response to TGF-β stimulation and beyond a clearly detectable base-line Tak1 phosphorylation level (Fig. 2-3C).

In Tak1/Wnt1-Cre cells, there was no detectable Tak1 phosphorylation, with or without TGF-β stimulation. A modest increase in phosphorylation of Smad-independent pathway targets p38

Mapk, Jnk, and Erk1/2 was induced by TGF-β2 in control cells, but in mutant cells no such change was observed (Fig. 2-3C). TGF-β2 stimulation induced C-terminal phosphorylation of both TGF-β Smads (Smad2) and BMP Smads (Smad1/5/8) in control palatal mesenchymal cells.

In mutant cells, Smad2 C-terminal also became phosphorylated in response to TGF-β2 stimulation, but less so than in control cells (Fig. 2-3, B and C), and BMP Smads did not show detectable C-terminal phosphorylation. In all samples, Smad2 protein levels remained stable for

90 min after TGF-β stimulation (Fig. 2-3B). Similar results were obtained using cells isolated from first pharyngeal arches (which also contribute to mandible and tongue) at E11, using a neural crest stem cell line in conjunction with the Tak1 kinase inhibitor (5Z-7-oxozeaenol) (Fig.

2-3F and data not shown).

In summary, the relative pattern of phosphorylation of downstream targets shown by control and mutant palatal shelf primary culture cells in response to TGF-β stimulation is similar to that found between control and mutant E14 palatal shelves recovered in vivo. Because Tak1 has also been shown to be involved in BMP signaling, we stimulated both the control and Tak1/Wnt1-Cre mutant ecto-mesenchymal cells with BMP2 and monitored the phosphorylation of signaling molecules as above (Fig. 2-3D). Unlike TGF-β2 and as expected,

BMP2 was not able to induce Tak1 phosphorylation at Ser-412 in control cells, nor did mutant cells show detectable phospho-Tak1 levels. Control cells were responsive to BMP2 stimulation

in their progressive increase in C-terminal BMP-Smad (Smad 1/5/8) phosphorylation, but Smad2 phosphorylation was not altered. In mutant cells, base-line C-terminal Smad1/5/8 phosphorylation levels were lower, and their responsiveness to BMP2 stimulation was reduced, when compared with control cells. Collectively, the results of these experiments suggest that in neural crest-derived mesenchymal cells, Tak1 contributes more to TGF-β than BMP-induced C- terminal phosphorylation of the corresponding R-Smads. BMP2 induced progressive p38 Mapk and Jnk phosphorylation in controls cells, but no increase was detected in mutant cells.

To provide additional evidence for the role of Tak1 in C-terminal phosphorylation of

Smad2, we transduced palatal mesenchymal cells carrying the homozygous Tak1-flox allele with recombinant adenoviruses expressing the Cre recombinase and analyzed the transduced and control cells (transduced with AdGfp) for Tak1 and Smad2 phosphorylation (Fig. 2-3E). These results confirm that the reduction in C-terminal phosphorylation of Smad2 is an immediate result of Tak1 deficiency rather than just a long term adaptation to severely reduced Tak1 levels in ourTak1/Wnt1-Cre mutants.

To conclude, we show that Tak1 is required for appropriate activation of both canonical and noncanonical TGF-β and BMP signaling pathways in neural crest-derived mesenchymal cells. Moreover, in the presence of Tak1, TGF-β2 was able to induce activation of both TGF-β and BMP R-Smads, i.e. Smad2 and Smad1/5/8, respectively.

Figure 2-3. Activation of R-Smads and Mapks is less in Tak1/Wnt1-Cre embryos. A, representative immunoblot from prefusion palatal shelves harvested from control and Tak1/Wnt1-Cre mutant embryos at E14.0 and analyzed for phosphorylation of Tak1, Smad1/5/8 (BMP Smads), Smad2 (TGF-β Smad), and p38 Mapk. Co, control sample; Mu, Tak1/Wnt1-Cre mutant sample. The bar graph shows relative quantification of pTak1, p38 Mapk, pSmad2-C, and pSmad1/5/8-C in controls (blue columns) and mutants (red columns) (normalized to β-actin; n = 4). B, primary palatal mesenchymal cell cultures were established (see “Experimental Procedures”) from prefusion palatal shelves (at E14.0) of mouse embryos carrying both the Wnt1-Cre transgene and the R26Rlacz reporter (top left). Cells stained blue are derived from the neural crest. Theblack arrows point to a few negatively staining cells. Scale bar, 200 µm. A representative immunoblot of primary palatal mesenchymal cells stimulated with TGF-β2 and analyzed for C-terminal Smad2 phosphorylation is shown. Top right, the diagram illustrates the differences in amount of normalized Smad2-C phosphorylation between control (blue data points and blue line) and Tak1/Wnt1-Cre mutant (red data pointsand red line) cells. The values were normalized to Smad2. C, representative immunoblot from primary palatal mesenchymal cells stimulated with TGF-β2 (10 ng/ml), and protein lysates were analyzed for Tak1, C-terminal Smad1/5/8, C-terminal Smad2, total Smad2, p38 Mapk, Jnk, and Erk1/2 phosphorylation. The bar graphs show relative quantification of pTak1, p38 Mapk, pSmad2-C, pJnk, and Smad2 in controls (blue columns) and mutants (red columns) (normalized

to β-actin; n = 3). D, representative immunoblot of primary palatal mesenchymal cells stimulated with BMP2 (100 ng/ml) and analyzed for Tak1 phosphorylation, C-terminal Smad1/5/8 phosphorylation, total Smad1, C-terminal Smad2 phosphorylation, total Smad2, p38 Mapk phosphorylation, and Jnk phosphorylation. The bar graphs show relative quantification of pTak1, p38 Mapk, pSmad1/5/8-C, and total Smad1 in controls (blue columns) and mutants (red columns) (normalized to β-actin; n = 3). Error bars, S.E. *, p < 0.05. E, representative immunoblot of primary Tak1FX/FX palatal mesenchymal cells transduced with AdGfp (control) or AdCre (mutant). The transduced cells were stimulated with TGF-β and analyzed for Tak1 phosphorylation, C-terminal Smad2 phosphorylation, total Smad2, and β-actin. Moi, multiplicity of infection. F, representative immunoblot of O9-1 neural crest stem cells stimulated with TGF- β2 in the presence or absence of the Tak1 kinase inhibitor 5Z-7-oxozeaenol (5Z) and analyzed for C-terminal Smad2 phosphorylation, total Smad2, and β-actin.

2-4.4. Agonist-induced Linker Region Phosphorylation Is Affected in Palatal Mesenchymal

Cells Deficient in Tak1 Mutants

Several studies have shown that in R-Smads the intervening sequence that links the DNA binding domain and transcriptional domain, the so-called linker region, becomes phosphorylated by a set of divergent stimuli (including FGFs, EGFs, stress signals) via Map kinases, and also by members of the TGF-β superfamily (Massague et al., 2005; Wang et al., 2009; Wrighton and

Feng, 2008). This agonist-induced linker region phosphorylation (ALP) at T220 in Smad2 (T179 in Smad3) located immediately upstream of a PY motif is of particular interest, since it was recently suggested that, in addition to priming Smads for turnover, phosphorylation of this threonine residue is required for a full transcriptional activity of Smad complexes (Alarcon et al.,

2009; Aragon et al., 2011). Therefore, we stimulated both control and Tak1-deficient primary mesenchymal cells (from both the first pharyngeal arch and the palate) with TGF-β2 and analyzed phosphorylation of these key residues (Smad2-L(T220), Smad3-L(T179)) and of

Smad2-L(S250) using phospho-residue-specific antibodies. Our results demonstrate that in control cells TGF-β2 induced efficient phosphorylation of the Smad2 linker region at S250 and

T220, and at T179 in Smad3, while cells deficient in Tak1 failed to show comparable induction of ALP (Fig. 2-4A). As before, practically identical results were obtained using cells isolated from first pharyngeal arches at E11.0 (data not shown). Moreover, O9-01 neural crest stem cells showed similar increases in TGF-β2-induced linker region phosphorylation at Smad2-L(T220) and Smad3-L(T179), and that this induction was largely inhibited by 5Z-7-Oxozeaenol (Fig. 2-

4A, lower panel).

To investigate whether these in vitro results were reflective of palatal shelf mesenchyme in vivo, we prepared protein lysates directly from pre-fusion palatal shelves of control and

Tak1/Wnt1-Cre mutant embryos and analyzed them for the Smad2/3 linker region phosphorylation. Endogenous levels of Smad2 linker phosphorylation at T220 and S250 and

Smad3 phosphorylation at T179 were all significantly lower in mutant palatal samples than in those of controls (Fig. 2-4B).

It has previously been shown that in several established cell lines, e.g., HEK293T and

HaCaT, that T220 in the linker region of Smad2 is directly phosphorylated by nuclear cyclin- dependent kinases Cdks8 and 9 (Alarcon et al., 2009; Aragon et al., 2011). Therefore we examined the effect of Tak1 inactivation on subcellular localization of total Smad2 both on palatal mesenchymal cells (harvested at E14) (Fig. 2-4C) and on first pharyngeal arch mesenchymal cells (harvested at E11 – data not shown). In both cell types, TGF-β2-induced

Smad2 nuclear accumulation was effectively inhibited by the Tak1 kinase inhibitor (5Z-7-

Oxozeaenol), and by the TGFβRI kinase inhibitor (SB431542) (Fig. 2-4C, and data not shown).

Tak1 kinase inhibitor also inhibited both TGF-β2–induced Smad2-C-terminal and linker region phosphorylation at Smad2-L(T220), Smad3-L(T179) and Smad2-S250 (Fig. 2-5A). Certain

Tak1-mediated ALP were also dependent on the presence of the TGFβRI protein, and the kinase activity of TGFβRI, since both the Cre-mediated Tgfbr1 deletion and TGFβRI inhibition by the

SB431542 could attenuate TGF-β2-induced phosphorylation at Smad2-L(T220)/Smad3-L(T179)

(Fig. 2-5A, B). As expected, agonist-induced phosphorylation of Smad2 at the C-terminus was also inhibited by the physical loss of Tgfbr1 and by SB431542 (Fig. 2-5A, B). In palatal mesenchymal cells, flavopiridol (Cdk inhibitor) inhibited ALP at T220 and S250 (Smad2) (Fig.

2-5A), while UO126 (Erk1/2 & Jnk inhibitor) could effectively prevent TGF-β2-induced Smad2-

L(S250) phosphorylation, but could only weakly inhibit ALP at T220 (Fig. 2-5A). The effect of

SB202190 (p38 Mapk inhibitor) on phosphorylation at Smad2-L(T220) was very similar to that

of UO126 (data not shown). Our cell fractionation experiments confirmed that most of the

Smad2 molecules with TGF-β2-induced phosphorylation at T220 could be detected in the nucleus and that this phosphorylation was effectively inhibited by flavopiridol (Fig. 2-5C).

In summary, our results show that in craniofacial mesenchymal cells Tak1 deficiency lead to reduced agonist-induced C-terminal phosphorylation and decreased nuclear accumulation of R-Smads, and that agonist-induced linker phosphorylation of Smad2 both at T220 (which is at least partially mediated by Cdks) and at S250 (mediated by Erk1/2 and/or Jnk) was dependent on the functional Tak1 protein (Fig. 2-5D-E).

Figure 2-4. Tak1 is required for appropriate Smad2 nuclear localization and agonist- induced Smad2/Smad3 linker-region phosphorylation in palatal mesenchymal cells. A, upper panel, primary palatal mesenchymal cells were stimulated with TGF-β2. Shown are representative Western blots of protein lysates analyzed for Smad2 linker region phosphorylation at Thr-220 (pSmad2-L(T220)) and Ser-250 (pSmad2-L(S250)) and for Smad3 linker region phosphorylation at Thr-179 (pSmad3-L(T179)). The bar graphs show relative quantification of pSmad2-L(T220), pSmad3-L(T179), and pSmad2-L(S250) in controls (blue columns) and mutants (red columns) (normalized to β-actin; n = 3). A,lower panel, representative immunoblot of O9-1 neural crest stem cells stimulated with TGF-β2 in the presence or absence of the Tak1 kinase inhibitor 5Z-7-oxozeaenol (5Z) or vehicle (dimethyl sulfoxide, DMSO) and analyzed for Smad2 linker region phosphorylation at Thr-220 (pSmad2-L(T220)), Thr-179 (pSmad3-L(T179)), total Smad2, and β-actin. B, representative immunoblot of prefusion palatal shelves harvested from control (Co) and Tak1/Wnt1-Cre mutant (Mu) embryos at E14.0 and analyzed for linker region phosphorylation at Thr-220 (pSmad2-L(T220)) and Ser-250 (pSmad2- L(S250)) and for Smad3 linker region phosphorylation at Thr-179 (pSmad3-L(T179)). The bar graph shows relative quantification of agonist-induced linker region phosphorylation in controls (blue columns) and mutants (red columns) (normalized to β-actin; n = 3). C, representative images showing subcellular localization of Smad2 in unstimulated cells (upper left), cells stimulated with TGF-β2 (upper right), cells stimulated with TGF-β2 in the presence of the TGFβRI kinase inhibitor (SB431542 (SB), lower left), and cells stimulated with TGF-β2 in the presence of Tak1 inhibitor (5Z-7-oxozeaenol (5Z), lower right). Red, immunostaining for

Smad2; blue, nuclear counterstaining with DAPI. The bar graph shows quantification of nuclear localization of Smad2 (n = 3; error bars, S.E.).

2-4.5. Tak1 as a TGF-β Signal Transducer in Neural Crest-derived Facial Mesenchymal Cells

Previous studies have demonstrated that TGF-β-induced Tak1 activation is dependent on the presence of TGFβRI protein and is mediated by an adaptor protein Traf6 (Sorrentino et al.,

2008; Yamashita et al., 2008). However, whether or not this activation is dependent on the

TGFβRI kinase activity is still controversial (Sorrentino et al., 2008; Yamashita et al., 2008). As shown above, the presence of either TGFβRI or its kinase activity is sufficient for agonist- induced Tak1-mediated linker region phosphorylation of Smad2 in ecto-mesenchymal cells

(Fig.2-5A, B). To better define the relationship between Tak1 kinase-mediated and TGFβRI kinase-mediated transcriptional responses, we compared expression profiles of TGF-β2- responsive genes in primary palatal mesenchymal cells in the presence of inhibitors for either

TGFβRI kinase or Tak1 kinase using a genome-wide transcriptomic analysis (Fig. 2-6). Of TGF-

β2-responsive genes (>2-fold change; p<0.05), 58 were dependent on TGFβRI kinase, 27 were dependent on Tak1 kinase and 18 were dependent on both (>2.5-fold change; p<0.05) (Fig. 2-

6A). Among the up-regulated genes (120 genes; >2-fold change; p<0.05), 47 were TGFβRI kinase-dependent, 20 were Tak1 kinase-dependent and 12 were dependent on both (>2.5-fold change; p<0.05) (Fig. 2-6B). Among the down-regulated (45 genes; >2-fold change; p<0.05) 11 were TGFβRI kinase-dependent, 7 were Tak1 kinase-dependent and 6 were dependent on both

(>2.5-fold change; p<0.05) (Fig. 2-6C). Differential expression of several Tak1 kinase/TGFβRI kinase-dependent genes was confirmed by real-time RT-PCR (Fig. 2-5D). When more stringent criteria were used and only the genes that displayed >3-fold expression change when stimulated with TGF-β2 were analyzed, 10 genes were dependent on both TGFβRI and Tak1 kinase

activities, while 8 genes and 1 gene were dependent on only TGFβRI or only Tak1 kinase activities, respectively (data not shown).

To examine whether any of the kinase-dependent gene expression findings reflected those between control and Tak1-deficient cells in vivo, we harvested NC-derived craniofacial mesenchymal tissues (both mandibular arch at E11 and palatal shelves at E14) from control and

Tak1/Wnt1-Cre mutant embryos and compared expression levels of TGF-β-responsive genes using real-time RT-PCR (Fig. 2-6E). Among the analyzed genes, Nfatc2, which was induced by

TGF-β2 in cultured palatal mesenchymal cells dependent on both kinases, was clearly down- regulated in Tak1/Wnt1-Cre mutant first pharyngeal arch tissues at E11 and slightly decreased though not significantly in mutant palatal shelves at E14. Similarly, Osr1, which was repressed by TGF-β2 in cultured palatal mesenchymal cells dependent on both kinases, was expressed at higher levels both in Tak1/Wnt1-Cre mutant first pharyngeal arch (2.2-fold higher) and prefusion palatal shelf tissues (2.8-fold higher) than in corresponding control tissues. Any differences in average expression of other genes tested, e.g., Wnt11 and Bmp4, were relatively modest, but some still showed tendencies consistent with the findings made in mesenchymal cell culture assays in vitro.

To conclude, TGFβRI and Tak1 kinases mediated both distinct and overlapping transcriptional responses in palatal mesenchymal cells, when stimulated by TGF-β2. In this cell type, agonist-induced Tak1 activation could occur independently of TGFβRI kinase activity.

Figure 2-5. Smad2 linker-region at Thr-220 is phosphorylated by nuclear kinases in palatal mesenchymal cells. A, representative immunoblot of primary palatal mesenchymal cells stimulated with TGF-β2 in the presence of SB431542 (SB), 5Z-7-oxozeaenol (5Z), UO126 (UO1), and flavopiridol (Fla; 1 and 10 µm (asterisk)). Protein lysates were analyzed for Smad2 C-terminal phosphorylation, Smad2 linker region phosphorylation (at Thr-220 and Ser-250), and Smad3 linker region phosphorylation (at Thr-179). The bar graphs show relative quantification of pSmad2-C, pSmad-L(T220), pSmad3-L(T179), and pSmad2-L(S250) (normalized to β- actin; n = 3). Error bars, S.E. *, p < 0.05. B, representative immunoblot of primary palatal mesenchymal cells from Tgfbr1FX/FX embryos were transduced with Ad-Gfp or Ad-Cre, the cells were stimulated with TGF-β2, and protein lysates were analyzed for C-terminal Smad2 phosphorylation, p38 Mapk phosphorylation, and linker region phosphorylation at Smad2- L(T220) and at Smad3-L(T179). β-actin was loading control. C, palatal mesenchymal cells were stimulated by TGF-β2 in the presence and absence of flavopiridol. Cytosolic and nuclear lysates were analyzed for Smad2 linker region phosphorylation at Thr-220 (pSmad2-L(T220)) and for Smad3 linker region phosphorylation at Thr-179 (pSmad3-L(T179)). Antibodies against PSMA2 and histone H3 were used to assess the purity of cytosolic and nuclear fractions (middle and bottom panels). D, schematic representation of Smad2 depicting Thr/Ser residues phosphorylated by nuclear Cdks and Jnk/Erk in the linker region and by TGFβRI (RI) at the C terminus examined in this study. E, schematic summary illustrating the role(s) of Tak1 in phosphorylation of Smad2 (left side of diagram) in which Smad2 C-terminal phosphorylation

levels were greatly enhanced in response to TGF-β stimulation when Tak1 was present, as well asTgfbr1. Cdk-dependent phosphorylation of Thr-220 occurs in the nucleus. Phosphorylation of Ser-250 is Mapk-dependent but also inhibited by Cdk inhibitor, which may be due to some dependence on the prior phosphorylation of Thr-220 in the model of Aragón et al. (Aragon et al., 2011). Deletion ofTak1 results in reduced C-terminal phosphorylation, leading to a reduced nuclear accumulation of Smad2, and reduced linker region phosphorylation (right side of diagram), which would be expected to reduce effectiveness in gene regulation according to the model of Aragónet al. (Aragon et al., 2011).

Figure 2-6. Tak1 and TGFβRI mediate both distinct and overlapping gene responses in palatal mesenchymal cells. Total RNAs from unstimulated palatal mesenchymal cells, from cells stimulated with TGF-β2, from cells treated with the TGFβRI kinase inhibitor (SB431542 (SB), 10 µm, 60 min prior stimulation) and stimulated with TGF-β2 (2 h), and cells treated with Tak1 inhibitor (5Z-7-oxozeaenol (5Z), 2 µm, 60 min prior stimulation) and stimulated with TGF- β2 (2 h) were subjected to genome-wide transcriptomic analysis on the MOE 430A 2.0 microarray. A, Venn diagram showing a summary of the number TGFβRI kinase- and Tak1 kinase-dependent TGF-β2 responsive genes (>2-fold change; p < 0.05). B, summary of number of TGFβRI kinase- and Tak1 kinase-dependent genes up-regulated by TGF-β2 in palatal mesenchymal cells (five examples of genes are shown in each group). C, summary of number of TGFβRI kinase- and Tak1 kinase-dependent genes that are down-regulated by TGF-β2 (3–5 examples of genes in each group). # in B and C denotes references highlighting relevance of a listed gene either in TGF-β signaling or in craniofacial/neural crest biology. D, real time RT- PCR quantification of differentially expressed genes. Blue columns, no inhibitor, no TGF-β2; red columns, no inhibitor, stimulated with TGF-β2; green columns, 10 µm SB431542 60 min prior stimulation with TGF-β2; purple column, 2 µm 5Z-7-oxozeaenol 60 min prior to stimulation with TGF-β2. E, bar graph showing relative real time RT-PCR quantification of selected TGF-β- responsive Tak1-dependent genes (identified by the microarray screen) in the craniofacial mesenchyme. Blue columns, control; red columns, Tak1/Wnt1-Cre mutant (n = 3). Error bars, S.E. *, p < 0.05. 1st pharyngeal arch, first pharyngeal arch harvested at E11.0; palatal shelves, prefusion palatal shelves harvested at E14.0.

2-5. Discussion

Although Tak1, a member of the Map kinase-kinase-kinase subfamily, was originally shown to mediate TGF-β signaling pathways (Yamaguchi et al., 1995), its physiological role in

TGF-β superfamily signaling in vivo, particularly during embryogenesis, is still poorly elucidated. Here we show that Tak1 plays a critical non-redundant role in craniofacial development, and that, in embryonic neural crest-derived craniofacial cells, Tak1 is required not only for Smad-independent TGF-β superfamily signaling, but also for maximal ligand-induced

C-terminal and linker phosphorylation of R-Smads.

2-5.1. Role of Tak1 in craniofacial development

Previous studies have shown that TGF-β and Bmp signaling plays an essential role in the neural crest-derived palatal mesenchyme(Baek et al., 2011; Dudas et al., 2006; Ito et al., 2003).

Of particular note is a recent report which demonstrates that cleft palate in Tgfbr2/Wnt1-Cre mutants results from TGF-β2-induced activation of an alternative pathway via TGFβRI and

TGFβRIII which induces activation of Tak1 and hence p38 Mapk (Iwata et al., 2012a). This aberrant activation results in attenuated expression of Fgf9 and Pitx2 leading to reduced mesenchymal cell proliferation and cleft palate(Iwata et al., 2012b). These results raised the questions of what role neural crest-expressed Tak1 normally plays in craniofacial development, and in the activation of signaling pathway components downstream of TGF-β2 signaling.

Here we report that Tak1 is required for normal facial and mandibular growth and palatogenesis in vivo and that it mediates both Smad-dependent and Smad-independent TGF-β and Bmp signaling in neural crest cells in vitro. The overall craniofacial phenotypes of

Tak1/Wnt1-Cre mutants were relatively mild when compared to those of corresponding Tgfbr1,

Tgfbr2 or Bmpr1a mutants. Unlike in Tgfb receptor mutants, palatal mesenchymal cell proliferation in general was only slightly affected in Tak1 mutants, as we did not detect any significant differences at the time points examined. Although over-activation of Tak1 in the

Tgfbr2/Wnt1-Cre mutants led to a reduction in cell proliferation (Holm et al., 2011), removal of

Tak1 in our Tak1/Wnt1-Cre mutants resulted in moderate hypoplasia. As we could not detect increased apoptosis between embryonal days 11-14, generalized hyperplasia in NC-derived tissues as the result of the absence of Tak1 does not seem to occur.

Normal formation of the secondary palate requires distinct developmental events; not just formation and growth of two palatal shelves but their elevation, physical contact with one another, fusion, loss of epithelial seam and differentiation into cartilage and bone. Of these steps, shelf elevation and patterning (ruga formation) showed developmental delay in Tak1Wnt1Cre mutants. It was recently shown that a Turing-type reaction-diffusion mechanism establishes the normal pattern of rugae (Economou et al., 2012). Although the delay in ruga formation may indicate an intrinsic defect in palatogenesis, it also is possible that a failure of the tongue to descend disturbs the shelf tissue proportions, altering the distribution of signals for ruga formation, and slowing their increase in number. An essential role for Tak1 in palatal shelves themselves in the processes of palatal shelf elevation onwards is hard to define as these processes occurred indistinguishably in control and Tak1/Wnt1-Cre mutant heads in rolling culture in vitro when tongue and mandible were absent but their morphology looked different from normal palatal shelves in vivo (Figure 2-1B). This result, along with histological observations, suggests that the failure of the tongue to descend sufficiently in mutants most likely contributes to the cleft palate phenotype in vivo by physically impeding shelf elevation. Consistent with these

findings, Song et al. have recently shown that Tak1 plays an important role in tongue development by controlling Fgf10 expression (Song et al., 2013) .

At the protein level, Tak1/Wnt1-Cre mutant tissues showed reduced levels of activation of both Smad- independent and -dependent pathways rather than an upregulation of Smad- dependent pathway signaling. As our studies on primary culture cells also showed, Tak1 functions as a modulator of signal strength rather than an ‘on/off switch’ for the Smad- independent pathway in the NC-derived craniofacial mesenchyme. Our results do not exclude that altered TGF-β/Bmp-independent Tak1-mediated signaling in these cells may also contribute to the relatively mild phenotype.

2-5.2. Role of Tak1 in TGF-β-induced signaling events

Our studies of the dependence of TGF-β2-induced signaling on Tak1 in embryonic neural crest cells have revealed important details not only of phosphorylation of components of signaling pathways but of their cell type-dependent variation. We show that in NC-derived craniofacial mesenchymal cells Tak1 mediates both TGF-β and BMP-induced activation of their corresponding R-Smads. Shim et al showed that in chondrocytes Tak1 can phosphorylate the C- terminal residues of an R-Smad (Smad1) only as part of the Bmp signaling pathway (Shim et al.,

2009). Moreover, we show that in neural crest cells TGF-β stimulation is able to stimulate BMP

R-Smad activation directly, and that this process is, in part, mediated by Tak1. In fact, a recent study on established immortalized cell lines (of both epithelial, endothelial and mesenchymal origins) suggested that this unconventional TGF-β-induced Bmp R-Smad activation is required for a subset of critical TGF-β-induced cellular functions (Daly et al., 2008). While our studies show that this unconventional R-Smad activation takes place in primary NC-derived craniofacial

mesenchymal cells in vitro, it remains to be shown whether it plays a role in craniofacial ectomesenchyme during embryogenesis in vivo.

Linker regions in both TGF-β and BMP R-Smads are Ser/Thr-rich and are known to be phosphorylated by several different kinases, e.g., GSK, Mapks and Cdks, and it has been suggested that these post-translational modifications have both activatory and inhibitory regulatory functions in TGF-β signaling (Wrighton and Feng, 2008). Here we show that palatal and pharyngeal arch mesenchymal cells deficient in Tak1 show reduced agonist-induced linker region phosphorylation both at T220 and S250 in Smad2 in vitro, and that particularly T220 phosphorylation in the Smad2 linker region is reduced in neural crest cell-specific Tak1 mutants in vivo. Moreover, our results suggest that in NC-derived mesenchymal cells T220 and S250 are phosphorylated by Cdks and Mapks, respectively. These findings differ from those made in fibroblast and melanoma cell lines, which showed that Mapks rather than Cdks are the primary kinases responsible for the linker region phosphorylation (Cohen-Solal et al., 2011; Hough et al.,

2012). Therefore, there seems to be distinct cell type-specific differences between the potency of particular kinases phosphorylate the specific residues.

It was recently suggested that ALP at Smad2-L (T220) plays a role in the so-called

“action-turnover switch” function of R-Smads, and may be required both for maximal TGF-β- induced transcriptional activity and efficient termination of the signal in immortalized cell lines

(Aragon et al., 2011; Gao et al., 2009). Our present results show that, in primary craniofacial mesenchymal cells, Tak1, by functioning as an amplifier of C-terminal R-Smad phosphorylation, could have indirectly regulated ALP at Smad T220 as deficient Tak1 activity would result in reduced ligand-induced R-Smad activation, and hence decreased R-Smad nuclear accumulation, and less linker-region phosphorylation at T220 by nuclear CDKs (Fig. 2-5E). However, our

results do not exclude the possibility that Tak1 could also contribute to linker region phosphorylation by other mechanisms, such as regulating activity of other downstream kinases

(Fig. 2-5E). Endogenous levels of Smad2-C phosphorylation and Smad T220 phosphorylation, were also reduced in Tak1/Wnt1Cre mutant palatal shelves, implying that Tak1 may modulate

TGF-β superfamily signaling in vivo in the same way.

2-5.3. Interdependence of TGFβRI and Tak1 Signaling?

Two recent studies addressed the mechanism of TGF-β-induced Tak1 activation in HEK

293 cells (Sorrentino et al., 2008; Yamashita et al., 2008). While these studies agree that a ligand-induced interaction between TGFβRI and Traf6 results in Traf6 auto-ubiquitination and subsequent Tak1 activation, they did not agree on a role for TGFβRI kinase activity in TGF-β- induced Tak1 activation. Here we show that, in craniofacial mesenchymal cells, the presence of

TGFβRI or its kinase activity are sufficient for Tak1-mediated signaling events downstream of

TGF-β2. To explore whether all the Tak1-dependent TGF-β transcriptional responses are dependent on the TGFβRI kinase activity we performed a genome-wide transcriptomic analysis on prefusion palatal mesenchymal cells stimulated with TGF-β2 in the presence or absence of either TGFβRI or Tak1 kinase inhibitors. This assay demonstrated that many of the TGF-β- induced transcriptional responses (both stimulatory and repressive) are simultaneously dependent on both TGFβRI kinase and Tak1 kinase activities, and that surprisingly few genes, e.g., Mllt3, respond to TGF-β2 stimulation if TGFβRI kinase activity is inhibited.

In conclusion, our combined evidence implies that Tak1 has a novel multi-modal role in the craniofacial neural crest-derived mesenchyme in regulating activation of both TGF-β- and

Bmp-induced Smad-dependent and Smad-independent signaling processes. Tak1 deficiency in

NC-derived mesenchymal cells leads to attenuation of both canonical and non-canonical TGF-β and Bmp signaling, which contribute to relatively subtle but consistent growth distortions in craniofacial structures. This dis-coordination in craniofacial growth likely results in delayed elevation of palatal shelves, which will never form a contact in the midline, resulting in cleft secondary palate.

Chapter 3:

Tak1, Smad4 and Trim33 cooperatively mediate TGF-β3 signaling during palate

development

3-1. Summary

Transforming growth factor-beta3 (TGF-β3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad- independent pathways work redundantly to transduce TGF-β3 signaling in this context.

However, detailed mechanisms by which this signaling is mediated still remain to be elucidated.

Here we show that TGF-β activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-β signaling, is functionally redundant with Smad4. Unlike the epithelium-specific Smad4 mutants, epithelium- specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-β signal transduction. Moreover, we show that inactivation of Tak1

in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-β signaling during palatogenesis and demonstrate that redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.

3-2. Introduction

Cleft palate, one of the most common birth defects in humans, is caused by a failure in palatogenesis (Chai and Maxson, 2006). During mammalian development, the secondary palate, which separates the oral cavity from the nasal cavity, develops as bilateral outgrowths (palatal shelves) of the maxillary processes of the first pharyngeal arch (Bush and Jiang, 2012). Palatal shelves first grow vertically down along the sides of the tongue, then they rapidly elevate and fuse in the midline (Ferguson, 1987). Failure in any of these three processes can result in cleft palate. The palatal shelf growth and patterning are governed by complex interactions between the ectoderm-derived epithelium, and the underlying mesenchyme derived from the cranial neural crest. One of the later events in palatogenesis, albeit a critical one, is epithelial fusion. During this process the medial edge epithelium (MEE) in tips of the apposing palatal shelves first forms the midline seam, which subsequently disappears. Many studies, both in humans and mice, have shown that signaling initiated by TGF-β3 is required for successful epithelial fusion (Dudas et al., 2007).

TGF-β3 binds and activates a heterotetrameric receptor complex composed of two type II and two type I receptors. Ligand-receptor interactions result in phosphorylation of TGF-β R-

Smads 2 and 3 and subsequent complex formation with a common Smad (Co-Smad or Smad4).

R-Smad/Co-Smad complexes then accumulate in the nucleus, where they function as

transcriptional co-regulators (Shi and Massague, 2003). This so called canonical (or Smad- dependent) signaling is regulated by many proteins interacting either with receptor complexes or

Smads, e.g., SARA, Axin, inhibitory Smads (I-Smads) and Trim33. Previous studies have shown that Trim33 (Tif1γ, ectodermin) may regulate TGF-β superfamily signaling in different ways depending on the biological context. Dupont et al. suggested that Trim33 is a negative regulator of TGF-β signaling by functioning as a monoubiquitin ligase capable of disrupting activated R-Smad/Co-Smad complexes , while He et al. showed that Trim33 can bind to activated R-Smads in competition with Smad4 and mediate distinct TGF-β signaling processes

(He et al., 2006). It also has been suggested that Trim33/R-Smad complexes can function as chromatin readers by making target genes accessible to R-Smad/Co-Smad complexes or by controlling the time the Smad complexes are bound to promoter sequences . In addition to the canonical (Smad-dependent) pathway, TGF-βs can also trigger non-canonical (Smad- independent) signaling processes leading to activation of various downstream mediators, such as, small rho-related GTPases, TGF-β-activate kinase-1 and downstream map kinase cascades including p38 Mapk, and Ikk-α (Derynck and Zhang, 2003).

Tgfb3 is strongly and specifically expressed in the MEE , and mouse embryos deficient in

Tgfb3, as well as epithelium-specific Tgfbr1 or Tgfbr2 mutants suffer from defective palatal epithelial fusion (Dudas et al., 2006; Xu et al., 2006). Xu et al. previously showed that, at least in palatal explant cultures in vitro, Smad-dependent (Smad4-mediated) and Smad-independent

(p38Mapk-mediated) pathways act redundantly during palatal epithelial fusion. Here, we show that redundancy between different TGF-β downstream mediators is even more extensive. By using tissue-specific mouse mutants in conjunction with a whole-head roller culture assay we demonstrate that both Tak1:Smad4- and Trim33:Smad4-double conditional mutants display

specific palatal fusion defects, and that simultaneous deletion/inactivation of all three proteins in palatal epithelial cells results in palatal phenotypes typically seen in tissue-specific Tgfb3 mutants.

3-3. Experimental Procedures

3-3.1. Mice

Tgfb3FX, IkkaFX and Trim33FX mice have been described earlier (Doetschman et al., 2012;

Kim and Kaartinen, 2008; Liu et al., 2008) . K14-Cre and Smad4FX mice were obtained from S.

Millar(Andl et al., 2004) and C. Deng (Yang et al., 2002), respectively. To generate mutant embryos, Cre-positive male mice heterozygous for floxed gene(s) were crossed with female mice carrying corresponding homozygous floxed allele(s) (see Table 3-1). For timed matings, the presence of a vaginal plug was designated as embryonic day 0 (E0). DNA for genotyping was prepared from tail tissues using DirectPCR lysis reagents (Viagen Biotech). Mouse lines were maintained in mixed genetic backgrounds. All experiments involving the use of animals were approved by the Institutional Animal Use and Care Committee at the University of Michigan-

Ann Arbor (Protocol #00004320).

3-3.2. Histology, Immunohistochemistry and cell death assays

Embryos were collected into sterile DPBS and fixed at 4°C overnight in freshly prepared

4% paraformaldehyde in PBS. Samples used for wax embedding were washed, dehydrated through a graded ethanol series (20, 50, 70, 95 and 100%) and an overnight step in 50%

Ethanol/50% Toluene, one hour step in 100% toluene, one hour step in 50% toluene/50% fresh

Blue Ribbon Tissue Embedding/Infiltration Medium (Leica Surgipath) before being oriented and

embedded in fresh Blue Ribbon Tissue Embedding/Infiltration Medium (Leica Surgipath) after three changes. 7µm sections (histology and immunohistochemistry) were cut, mounted on

Superfrost plus slides (Fisher) and stained with hematoxylin and eosin according to standard protocols. For immunohistochemistry, sections were rehydrated, and after antigen retrieval (5-20 minutes at 95-100°C in 10 mM citrate buffer, pH6.0) proliferating cell were detected using Ki67 antibody (#M7249; Dako). Cells positive for phosphorylated p38 Mapk were detected using p- p38 Mapk antibody (#4511; Cell Signaling). Antibody binding was visualized with Alexfluor-

594 secondary antibody (Life Technologies). Apoptotic cells were detected using a TUNEL assay (Dead End, Promega) following manufacturer’s instructions. Fluorescent images were viewed on an inverted fluorescent Leica DMI3000B microscope and documented using an

Olympus DP72 camera.

Table 3-1. Crosses used to generate mutant embryos

Male mouse Female mouse

Tgfb3FX/+K14Cre Tgfb3FXFX

Smad4FX/+K14Cre Smad4FXFX

Tak1FX/+Smad4FX/+K14Cre Tak1FXFXSmad4FXFX

IkkaFX/+Smad4FX/+K14Cre IkkaFXFXSmad4FXFX

Trim33FX/+Smad4FX/+K14Cre Trim33FXFXSmad4FXFX

3-3.3. In situ hybridization

Embryos were processed and embedded in paraffin as described above. 10µm sections were cut and mounted on glass slides. RNA probes were labeled using a DIG-labeled NTP mix

(Roche Applied Science) according to manufacturer’s instructions, stored at -80°C and diluted in hybridization buffer. Section ISH was performed as described (Moorman et al., 2001). After staining, sections were fixed and mounted in Immumount (Thermo Scientific). Probe templates for Tgfb3 and Mmp13 were prepared as described .

3-3.4. Real-time quantitative PCR

Tissues were harvested from tips of palatal shelves from E14 and E15 mouse embryos, placed into 200µl of RLT (RNeasy mini kit, Qiagen), and RNAs were isolated by using RNeasy columns (Qiagen). cDNAs were synthesized by using Omniscript reverse transcriptase (Qiagen) according to the manufacturer’s protocols. Real time quantitative PCR experiments were done either by using Universal Probe library-based assays (Roche Applied Science) or by using

TaqMan assay reagents (Applied Biosystems) (see Table 3-2). 30µl assays were quantified using

Applied Biosystems ABI7300 PCR and ViiA7 detection systems and software.

Table 3-2. Real-time quantitative PCR primers

Q-RTPCR using the Universal Probe System (Roche) Gene Forward Sequence Reverse Sequence Universal Probe Cytokeratin 14 (K14) atcgaggacctgaagagcaa tcgatctgcaggaggacatt #83 Tp63 (delta N-p63) ggaaaacaatgcccagactc aatctgctggtccatgctgt #45 Cdkn1a (p21) cagatccacagcgatatcca ggcacactttgctcctgtg #21

Q-RTPCR using the TaqMan approach (Life Technologies) Gene TaqMan Reference Irf6 Mm00516797_m1 Mmp13 Mm00439491_m1 Cdkn2a (p16) Mm00494449_m1 Cdkn1c (p57) Mm01272135_g1

3-3.5. Whole-head roller culture assays

Roller culture assays were modified from Goudy et al. (Goudy et al., 2010) Heads from

E15 embryos were collected in DPBS and mandibles, tongues, and brains were removed. The resulting mid-face samples were cultured for 24 hours at 37°C in roller bottles (60ml serum bottles) (60 rotations/minute) in serum free BGJb medium without penicillin and streptomycin.

The bottles were gassed at the beginning of the culture and again every 12 hours by gently bubbling the medium for 2 minutes with 02/CO2 (95%/5%). The palatal cultures were fixed, sectioned and stained as described above. In 5Z-7-oxozeanenol (Tak1 inhibitor; 10µM; eMolecules) treated samples, the inhibitor was added directly into the culture medium.

3-3.6. Western-blot assays

Palatal shelf edges were removed and cultured as described above in 2ml cryovials (60 rotations/minute) at 37°C in BGJb medium for 1 hour and then stimulated with TGF-β3

(10ng/ml) (#243-B3-002; R&D Systems) for 40 minutes. Tissues were then isolated and lysed in

2x Laemmli sample buffer (Harlow and Lane, 1988), ran on NuPage 4-12% Bis-Tris gradient gels (Invitrogen) and transferred by “iBlot dry blotting” (Invitrogen) onto nitrocellulose filters.

Immunobloting and detection were done using standard protocols. The antibodies used were p-

38 (N-20) (#SC-728; Santa Cruz Biotechnology) and p-p38 MapK (#4511; Cell Signaling).

3-3.7. Statistical analysis

For histological and real-time quantitative PCR analyses three or more samples were analyzed. Averages, standard error and probability (Student’s t-test, 2-tailed) were calculated.

Probability (p) equal or less than 0.05 was marked as statistically significant.

3.4. Results

3-4.1. Epithelium-specific Smad4 mutants display mild defects in palatogenesis

Smad-dependent and Smad-independent pathways have been shown to play functionally redundant roles during palatal epithelial fusion (Iwata et al., 2013) . To confirm and expand these findings, we first compared palatal phenotypes of epithelium-specific Tgfb3 mutants

(Tgfb3F/FK14-Cre = Tgfb3-cKO) to those of corresponding Smad4-cKOs (Smad4F/F:K14-Cre).

Tgfb3-cKOs display a fusion defect in the anterior palate, failure of the anterior palate to fuse to the nasal septum, variable degree of fusion in the mid-palate, persistent midline seam and occasionally a cleft in the posterior palate (Fig. 3-1 A-F). These observed phenotypes are less severe than those seen in Tgfb3 germline mutants , but are practically identical to those seen in epithelium-specific Tgfbr1 and Tgfbr2 mutants suggesting that the phenotypic difference between Tgfb3 germ line mutant and Tgfb3-cKO results, at least in part, from the inability of

K14-Cre to recombine in the periderm as shown by ((Lane et al., 2014); Chapter 4). In contrast to phenotypes seen in Tgfb3-cKOs, palatal phenotypes displayed by Smad4-cKOs were very mild (Fig. 3-1G-I, M and Table 3-1), yet consistent and previously unappreciated . These include posterior epithelial triangles and occasional failure of the anterior palate to fuse, and the nasal septum to fuse to the anterior palate (1/4).

3-4.2. Simultaneous deletion of TGF-β activated kinase-1 and Smad4 results in anterior and posterior palate defects

To further examine cooperation between Smad-independent and Smad-dependent pathways during palatal epithelial fusion in vivo, we generated an allelic series of double mutants lacking one or both alleles of Tak1 (which, at least in some cell and/or tissue types, is an upstream activator of p38 Mapk ) and Smad4 in epithelial cells by crossing transgenic K14-Cre male mice that also were heterozygous for both Tak1FX and Smad4FX alleles with double homozygote Tak1FXFXSmad4FXFX female mice (Table 3-3). Epithelium-specific double heterozygotes showed no palatal phenotypes, while corresponding Tak1:Smad4 double mutants

(herein called Tak1:Smad4cdKO) displayed specific and consistent defects in palatogenesis (Fig.

3-1J-L, M, and Table 3-1), e.g., persistent epithelial seam, anterior fusion defect and a failure of the nasal septum to fuse to the anterior secondary palate. Cre-positive embryos heterozygous for the floxed Tak1 allele and homozygous for the floxed Smad4 allele showed similar but milder defects as dcKO with variable penetrance (Table 3-3).

Figure 3-1. Epithelium-specific deletion of Tak1 and Smad4 results in defects in palatogenesis. Frontal sections on the anterior level (A, D, G, J), mid anterior (B, E, H, K) and posterior (C, F, I, L) levels (see N for levels of sections along the anterior-posterior axis). A-C, Control; D-F, Tgfb3-cKO, G-I, Smad4-cKO, J-L, Tak1:Smad4-dcKO. Asterisks in A, B, C, G illustrate the palatal mesenchymal confluence, black arrows in D and J point to the sites of failed fusion in the anterior palate, black arrows in E and K point to a gap between the nasal septum and the secondary palate, the black arrow in F points to an epithelial bridge in the posterior palate and black arrowheads in H, I, K and L point to persistent epithelial seams. Scale bar, 200 µm. Bar graph (M) illustrates the degree of fusion (mesenchymal confluence); Error bars, SEM; *, p<0.05; Ant, anterior level; Post, posterior level; NS, fusion between the nasal septum and secondary palate. N, mouse mouth roof at E18; superior view; red lines illustrate the levels of frontal sections shown in A-L (n=3).

Table 3-3. Summary of palatal defects in Tgfb3, Smad4, Tak1 cKOs and Tak1:Smad4 –dcKOs.

Genotype Anterior cleft/ Nasal septum Posterior Posterior cleft persistent fusion defect/ midline seam/ seam persistent epithelial seam triangle

Control (Cre-); n=3 0/3 0/3 0/3 0/3 Tgfb3FxFxK14Cre; n=3 3/3 3/3 2/3 1/3 Smad4FXFXK14Cre; n=4 1/4 1/4 4/4 0/4 Tak1FXFXK14Cre; n=3 0/3 0/3 0/3 0/3 Tak1FX/+Smad4FXFXK14Cre; 1/3 2/3 2/3 0/3 n=3 Tak1FX/+Smad4F/+K14Cre; 0/3 0/3 0/3 0/3 n=3 Tak1FXFXSmad4FXFXK14Cre; 4/4 4/4 4/4 1/4 n=4

3-4.3. Additional modifiers of TGF-β signaling during palatal epithelial fusion

Our results suggested that Tak1 plays a redundant regulatory role in TGF-β signaling, perhaps by functioning upstream of p38 Mapk in palatal epithelial cells. Yet, the Tak1:Smad4- dcKO palatal phenotypes (Fig. 1J-L) were noticeably less severe than those seen in corresponding Tgfb3-cKOs (Fig. 1D-F) or Tgfbr1-cKOs (Dudas et al., 2006)). Therefore we wondered whether, in addition to the Tak1-mediated pathway, other signal mediators could be involved in TGF-β3-induced palatal epithelial fusion. As candidates, we chose Ikk-α (Chuk) and

Trim33 (Tif1γ; ectodermin), because both proteins modulate TGF-β signaling both by Smad- dependent and Smad-independent mechanisms contingent on the biological context (Descargues et al., 2008; Xi et al., 2011). Our results showed that epithelium-specific single mutants (i.e.,

Ikka-cKOs and Trim33-cKOs) did not show palatal phenotypes, and that Ikka:Smad4-dcKO mutants did not significantly differ from those of Smad4-cKOs (Fig. 2A-F,M) suggesting that

Ikk-α does not contribute to TGF-β-triggered MEE removal. However, Trim33:Smad4-dcKO mutants consistently showed the persistent midline epithelial seam both in the anterior and in the posterior palate, and between the nasal septum and the anterior palate (Fig. 2G-L,M and Table

IV). This phenotype differs from that seen in Smad4-cKOs or in Tak1:Smad4-dcKOs (compare

Fig.1 and 2) suggesting that Trim33 and Smad4 play functionally redundant roles in palatal epithelial fusion that are at least in part distinct from that of a combined role of Tak1 and Smad4.

Figure 3-2. Simultaneous deletion of Trim33 and Smad4 in epithelial cells leads to persistence of the midline seam. Frontal sections on the anterior level (A, D, G, J), mid anterior (B, E, H, K) and posterior (C, F, I, L) levels (see Fig 1N). A-C and G-I, Control; D-F, Ikka:Smad4-dcKO, J-L, Trim33:Smad4-dcKO. Asterisks in A, B, C, D, E, G, H, I illustrate the palatal mesenchymal confluence, black arrowheads in E point to epithelial islands between the nasal septum and the posterior palate, black arrows in F, J, K, and L point to persistent epithelial seams. Scale bar, 200 µm. Bar graph (M) illustrates the degree of fusion (mesenchymal confluence); Error bars, SEM; *, p<0.05; Ant, anterior level; Post, posterior level; NS, fusion between the nasal septum and secondary palate (n=3).

Table 3-4. Summary of palatal defects in Ikka and Trim33 cKOs and Ikka:Smad4 and

Trim33:Smad4-dcKOs.

Genotype Anterior cleft/ Nasal septum Posterior Posterior cleft persistent fusion defect/ midline seam/ seam persistent epithelial seam triangle Control (Cre-); n=3 0/3 0/3 0/3 0/3 IkkaFFK14Cre; n=3 0/3 0/3 0/3 0/3 Trim33FFK14Cre; n=3 0/3 0/3 0/3 0/3 IkkaFXFXSmad4FXFXK14Cre; 0/3 1/3 2/3 0/3 n=3

3-4.4. Expression of a TGF-β signaling target Mmp13 is affected in Tak1:Smad4 and

Trim33:Smad4 double conditional mutants

It has been previously shown that Mmp13 expression is rapidly induced by TGF-β3- signaling in the midline seam during palatogenesis (Dudas and Kaartinen, 2005). To analyze whether Tak1 and Smad4- or Trim33 and Smad4-mediated pathways are involved in regulation of Mmp13 expression, we first harvested palatal shelves from control, Tgfb3-cKO, Smad4-cKO,

Tak1:Smad4-cKO and Trim33:Smad4-cKO embryos at E15.0 and used qRT-PCR to quantify their Mmp13 mRNA levels. As shown in Fig. 3A, Mmp13 expression was significantly reduced in Tgfb3, Tak1:Smad4-dcKO and Trim33:Smad4-dcKO samples, while the reduction in Smad4- cKO samples was rather modest and not statistically significant (Fig. 3A). We also used section

ISH to analyze Mmp13 and Tgfb3 expression in the MES and nasal septum epithelium (Fig. 3B-

I). Cre-negative control and Smad4-cKO samples displayed strong Tgfb3 and Mmp13 expression both in the MES and in the nasal septal epithelium. In Tak1:Smad4-cdKOs there was a notable reduction in Mmp13 expression, while in Trim33:Smad4-cKOs Mmp13 expression was barely detectable. In both samples, Tgfb3 mRNA levels did not differ from those seen in controls. These results suggest that a known TGF-β signaling target Mmp13 is regulated both by Tak1:Smad4- and Trim33:Smad4-mediated pathways in palatal medial edge epithelial cells.

3-4.5. Altered gene expression in TGF-β pathway mutants

Iwata et al. recently reported that signaling via TgfβRII stimulates Irf6 expression, which in turn suppresses dNp63 expression allowing appropriate p21 (Cdkn1a, p21Cip1) expression .

In prefusion palatal shelves at E14, we could not detect any differences in Irf6 or dNP63 expression levels between the control, Tgfb3-cKO, Smad4-cKO, Tak1:Smad4-cKO or

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Tak1:Smad4-dcKO samples (Fig.3J). In the fusing palatal shelves at E15, Irf6 was reduced only in Tak1:Smad4-dcKO samples, while dNP63 levels were increased in Tgfb3- and Smad4-cKO samples. In contrast to the lack of differences in Irf6 and dNP63 expression at E14, p21 expression levels were reduced in all samples lacking Smad4 (i.e., Smad4-cKO, Tak1:Smad4- dcKO and Trim33:Smad4-dcKO),but not in Tgfb3-cKO samples (Fig. 3J). In addition to p21,

TGF-β signaling has been shown to regulate expression of other cyclin-dependent kinase inhibitors in the MEE . Therefore, we compared expression levels of p15 (Cdkn2b, p15Ink4b), p16 (Cdkn2a, p16Ink4a) and p57 (Cdkn1c, p57Kip2) as outlined above (Fig. 3K and data not shown). At E14, p16 expression was significantly reduced in Tgfb3-cKOs and in Tak1:Smad4- dcKOs, but not in Trim33:Smad4-dcKOs, whereas Tgfb3-cKO samples showed a dramatic increase in p57 expression at E15 (expression levels of p15 were very low and did not show detectable differences between controls and different mutants; data not shown). Taken together, differences in expression level of candidate genes between Tak1:Smad4-dcKOs and Tgfb3-cKOs suggest that Tak1 and TGF-β3 can affect at least in part distinct pathways that act together to promote palatal epithelial fusion. Similarly, differences in gene expression between

Trim33:Smad4 and Tak1:Smad4 double mutants imply that these molecules mediate distinct arms of TGF-β signaling.

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Figure 3-3. Gene expression differences in prefusion and fusing palatal shelves between controls and TGF-β pathway mutants. A, QRT-PCR analysis of Mmp13 expression in palatal tissues of control (blue), Tgfb3cKO (red), Smad4-cKO (green), Tak1:Smad4-dcKO (purple) and Trim33:Smad4-dcKO (turquoise) at E15 (Error bars, SEM; *, p<0.05, n=3). B-I, In situ hybridization for Tgfb3 (B, C, D, E) and for Mmp13 (F, G, H, I). B, F, Control; C, G, Smad4- cKO; D, H, Tak1:Smad4-dcKO; E, I, Trim33:Smad4-dcKO. Frontal sections of E15 embryos; anterior level. Scale bar in I, 100µm (for B-I). J, QRT-PCR analysis of Irf6, dNP63 and p21 expression in palatal tissues from control, Tgfb3-cKO, Smad4-cKO, Tak1:Smad4-dcKO and Trim33:Smad4-dcKO embryos harvested at E14 and E15 (Error bars, SEM; *, p<0.05, n=3). K, QRT-PCR analysis of p16, and p57 expression in palatal tissues from control, Tgfb3-cKO, Smad4-cKO, Tak1:Smad4-dcKO and Trim33:Smad4-dcKO embryos harvested at E14 and E15 (Error bars, SEM; *, p<0.05; n=3).

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3-4.6. Epithelial cell proliferation and apoptosis are variably affected in Tak1:Smad4- and

Trim33:Smad4-dcKOs

Previous studies showed that TGF-β signaling in the palatal epithelium induces cell cycle arrest that is a prerequisite for the disappearance of the midline seam by apoptosis and other possible mechanisms . To examine whether cell proliferation in the MES was affected in

Tak1:Smad4- or Trim33:Smad4-dcKOs, we used Ki67 immunostaining on anterior frontal sections obtained from control (cre-negative) and dcKO embryos at E14.5 (Fig. 4A-C). Both

Tak1:Smad4 and Trim33:Smad4-dcKOs showed an increased number of positively staining epithelial cells in the tip of the nasal septum and in epithelial tips of prefusion palatal shelves when compared to the controls (Fig. 4A-C, J). In contrast, the number of TUNEL-positive apoptotic cells was significantly reduced in the control and mutant samples in the anterior palate on the level of nasal septum (Fig. 4D, F, H). MEE cells of Tak1:Smad4-dcKOs showed reduced phosphorylation (activation) of apoptosis-associated TGF-β-induced signaling molecule p38

Mapk, while Trim33:Smad4-dcKOs did not demonstrate comparable changes (Fig. 4 E, G, I).

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Figure 3-4. Proliferation and cell death are affected in Tak1:Smad4 and Trim33:Smad4-dcKOs. Immunostaining for Ki67 (red) in control (A), Tak1:Smad4- (B) and Trim33:Smad4-dcKO (C) at E14.5. Frontal sections, anterior level. Counterstaining with DAPI (blue nulei). White arrowheads in A-C point to Ki67-positive cells in the nasal septum epithelium. TUNEL staining to detect apoptotic/necrotic cells (white arrows in D, F, H; green nuclei) in control (D), Tak1:Smad4:dcKs (F) and in Trim33:Smad4-dcKO(H) samples. Frontal sections, anterior level; counter staining with DAPI (blue). Immunostaining for phosphorylated p38 Mapk (p-p38, red staining) in control (E), Tak1:Smad4:dcKs (G) and in Trim33:Smad4-dcKO (I) samples. White arrows in E, G, and I point to positively-staining cells. Frontal sections, anterior level; counter

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staining with DAPI (blue). J, Bar graph shows quantification of proliferation (Ki67) apoptosis (TUNEL) and phopho-p38 Mapk assays (Error bars, SEM; *, p<0.05; n=3).

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3-4.7. Inactivation of Tak1 in Trim33:Smad4-dcKOs phenocopies the palate defects seen in

Tgfb3-dcKOs

As outlined above, the palatal phenotypes of both Tak1:Smad4- and Trim33:Smad4- dcKOs, while consistent and clear, were still less severe than those seen in Tgfb3-cKOs.

Therefore, we wondered whether combined loss of Tak1, Smad4 and Trim33 would result in palatal phenotypes similar to those seen in Tgfb3-cKOs. To this end, we used a potent Tak1 inhibitor, 5Z-7-oxozeaenol, in conjunction with Trim33:Smad4-dcKOs in vitro by using a whole- head roller culture assay. Control cultures treated with 5Z-7-oxozeaenol, which efficiently blocked p38 Mapk phosphorylation (Fig. 3-5I), showed consistent and reliable fusion identical to that seen in untreated cultures (Fig. 3-5A-D). Furthermore, Smad4-cKO cultures with the Tak1 inhibitor did not demonstrate detectable phenotypes (data not shown). Under similar culture conditions, Trim33:Smad4-dcKOs without the Tak1 inhibitor showed a persistent midline seam both in the anterior and posterior palate, comparable to that seen in Trim33:Smad4-dcKOs in vivo (Fig. 3-5E,F). However, treatment of double-cKO cultures with the Tak1 inhibitor resulted in palatal phenotypes practically identical to those seen in Tgfb3-cKOs, i.e., an anterior hole and a posterior epithelial bridge (Fig. 3-5G,H). These data suggest that Tak1, Smad4 and Trim33 are redundantly transducing TGF-β3 signaling to remove the midline epithelial seam, and to facilitate palatal anterior and posterior fusion during palatogenesis (Fig. 3-6).

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Figure 3-5. Conditional removal of Trim33 and Smad4 along with Tak1 inhibition recapitulates the palatal phenotype seen in Tgfb3-cKO embryos.

A whole-head roller culture assay of Cre-negative controls (A-D) and Trim33:Smad4-dcKOs (E- H) in the absence (A,B, E, F) and presence of Tak1 inhibitor (C, D, G, H). A, C, E, G, frontal sections, anterior level; B, D, F, H, frontal sections, posterior level. Asterisks in A, B, C, D illustrate mesenchymal confluence, black arrow in D points to oral epithelial triangle, black arrows in E point to intact MEE cells in palatal shelves and nasal septum, black arrow in F points to the persistent epithelial seam, black arrows in G point to the anterior cleft and black arrow in H points to the posterior epithelial bridge. Scale bar, 100µm. I, Tak1 inhibitor 5Z-7-oxozeaenol (5Z) prevents p38 Mapk phosphorylation in palatal shelves harvested from whole-head roller cultures. J, bar graph shows a degree of palatal fusion in control and Trim33:Smad4-cKO samples cultured a presence or absence of 5Z in a whole-head roller culture assay. Error bars, SEM; *, p<0.05; n=3). Blue columns, anterior palate; red columns, posterior palate.

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Figure 3-6. Schematic of redundant functions of Tak1, Smad4 and Trim33 during palatal epithelial fusion.

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3-5. Discussion

Palatogenesis is a complex developmental process involving appropriate growth, elevation and fusion of palatal shelves (Ferguson, 1988). While the role of TGF-β3 in the palatal epithelium (MEE) is well established , recent studies have shown that mechanisms by which the downstream signaling is transduced are surprisingly complicated, involving redundantly acting canonical and non-canonical arms of TGF-β signaling . Here we show that in MEE cells, redundancy of TGF-β signal transduction is even more extensive, since recapitulation of the

Tgfb3-cKO palatal phenotype required inactivation of three separate genes/gene products (Tak1,

Smad4, Trim33) mediating different arms of TGF-β signaling.

Developing palatal shelves are composed of the mesenchyme (largely derived from the cranial neural crest with a minor contribution coming from the cranial paraxial mesoderm) and from the ectoderm-derived surface epithelium, which is covered by a thin layer of peridermal cells (Lane and Kaartinen, 2014). Recent studies have shown that peridermal cells play a key role in palatal shelf adherence, and that this layer has to be removed for a normal palatal adhesion and fusion to take place . Tgfb3 is expressed in both cell types (Lane et al., 2014) and interestingly, the cleft palate phenotype of Tgfb3 null mutants can be partially rescued by transducing Tgfb3 exclusively in peridermal cells . Likewise, Tgfb3-cKO mice, which lack Tgfb3 in epithelial cells including the MEE but not in peridermal cells, display significantly milder palatal phenotypes than those seen in systemic Tgfb3 mutants ((Lane et al., 2014) and the present study), suggesting that TGF-β3 signaling is important for peridermal cell removal and is thus required for adhesion and fusion between the palatal shelves in the mid-palate. In fact, Tgfb3- cKO palatal phenotypes are practically identical to those seen in epithelium-specific Tgfbr1 or

Tgfbr2 mutants . Moreover, this distinct palatal phenotype can be seen in mice expressing Tgfb1

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in the Tgfb3 locus (Yang and Kaartinen, 2007) suggesting that Tgfb1 can functionally replace

Tgfb3 in peridermal cells but not in MEE cells. Nevertheless, the consistent defects of Tgfb3- cKO embryos in the anterior and posterior palate (anterior hole, nasal septum fusion defect and posterior epithelial bridge/submucous cleft) show that TGF-β3-mediated signaling is crucial also in the MEE, and that the K14-Cre driver line is a useful tool to study gene functions in this cell type during palatogenesis.

A previous study on epithelium-specific Tgfbr2:K14-Cre mice suggested that TGF-β via

TgfbRII triggers a Smad4-Irf6 signaling cascade, which downregulates dNP63 resulting in p21 induction, cell cycle arrest and removal of MEE cells . Using the same K14-Cre transgenic line we demonstrate that Tgfb3-cKO embryos, which display clear and consistent palatal defects similar to those seen in Tgfbr2:K14-Cre mutants, do not show differences in p21 expression in prefusion or fusing palatal shelves. In addition, Smad4-dcKOs, which display a very mild phenotype in the posterior palate, consistently show reduced levels of p21 expression. Based on these results we conclude that changes in p21 expression are not causally related to the observed palatal phenotypes. Despite the obvious reduction in p21 expression in Smad4-cKOs as well as in

Tak1:Smad4- and Trim33:Smad4dcKOs, we could not see significant differences in Irf6 or dNP63 expression in prefusion palatal shelves at E14. In fusing palatal shelves at E15,

Tak1:Smad4-dcKOs palatal shelves showed reduction in Irf6 expression while palatal tissues harvested from Tgfb3-ckos at E15 showed an increase in dNP63. Whether these discrepancies between our current findings and those reported by others are caused by experimental variables, e.g., timing and/or technique of tissue harvest and differences in genetic backgrounds, or whether they are caused by other currently unknown causes, remains to be shown.

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In epithelial cells, TGF-βs are well-known growth inhibitory cytokines . It has been suggested that inhibition of MEE proliferation by TGF-β signaling triggers a cascade of events that results in the removal of MEE via apoptosis (and to some extent by transdifferentiation/migration) . Despite some differences in gene expression patterns between

Tak1:Smad4-dcKOs and Trim33:Smad4-dcKOs, we show that both of these epithelium-specific double mutants show increased levels of proliferation, particularly in the epithelial tips of the nasal septum, while increased levels of TUNEL-positive nuclei could be seen both in the epithelial tips of palatal shelves and nasal septum. Increased cell proliferation is consistent with our finding that p21 expression levels are reduced in both dcKOs. However, it is likely that expression of other negative cell cycle regulators, e.g., p16, is also regulated by TGF-β, as p21 expression levels are not reduced in Tgfb3 mutants, although they display a well-documented failure to suppress cell proliferation in the prefusion MEE .

A role of Tak1 in regulation of TGF-β3-induced p38Mapk activation is currently controversial. Several studies have suggested that Traf6-Tak1 signaling module mediates TGF-β- induced p38 Mapk activation , while other studies have proposed that instead of Tak1 other

Map3Ks, e.g., Map3K4 and Map3K10, play the key role in transducing TGF-β signaling that results in p38 Mapk phosphorylation . We previously showed that neural crest-derived ectomesenchymal cells lacking Tak1 fail to demonstrate TGF-β-induced p38 Mapk activation .

Our present results imply that also in MEE cells Tak1 is at least partially responsible for mediating p38 Mapk activation, since we could see reduction in p38Mapk phosphorylation in

Tak1:Smad4-dcKOs when compared to controls or Trim33:Smad4-dcKOs. Moreover, similar to palatal explant cultures with inactivated p38 Mapk , inactivation of Tak1 alone, which totally abolished the p38Mapk phosphorylation (Fig. 3-5I), did not result in defects in palatogenesis

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(Fig. 3-5), while simultaneous inactivation of Tak1 and deletion of Smad4 in explant cultures resulted in palatal phenotypes similar to that seen in Tak1:Smad4-dcKOs in vivo (Supplemental

Fig. 3-1).

Mutations in the IRF6 gene cause van der Woude syndrome, an autosomal dominant syndrome characterized by cleft lip and/or palate . Mouse embryos lacking Irf6 display early differentiation defects of the ectoderm, which are practically identical to those seen in Ikka knockout mice . Therefore, it is surprising that deletion of Ikka in epithelial cells including the

MEE does not result in noticeable defects in palatogenesis. Instead we discovered that in MEE cells, concomitant deletion of a novel chromatin reader Trim33 and Smad4 led to defective palatal fusion. Several recent reports have demonstrated that epigenetic mechanisms involving appropriate histone modifications are crucial for successful craniofacial development and palatogenesis. In humans, haploinsufficiency of histone-3 demethylases, e.g., PHF8 and KATB6, has been shown to result in cleft palate , while neural crest specific deletion of Hdac3 resulted in severe craniofacial defects including cleft palate in mouse embryos . Trim33 is a multifunctional protein , which has been shown to both facilitate and antagonize TGF-β signaling. In differentiating stem cells, Trim33, when complexed with Smad2, is able to bind to specific poised chromatin marks found in the promoter regions of master regulators, rendering them accessible to R-Smad/Co-Smad complexes (Xi et al., 2011). In other cellular systems, Trim33 has been shown to mono-ubiquitinate Smad4 disrupting Smad4-chromatin complexes and limiting their residence time at TGF-β-responsive enhancer s (Agricola et al., 2011). Here we show in MEE cells Trim33 does not function as a negative regulator of canonical TGF-β signaling, but rather that Trim33 and Smad4 function cooperatively during palatal fusion (Fig.

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3-6). A similar cooperative role of Smad4 and Trim33 in a control of neural stem cell proliferation was recently shown to take place in the developing cortex (Falk et al., 2013)

In conclusion, our data indicate that complex and largely cooperative mechanisms involving both non-canonical and canonical arms of TGF-β signaling control disappearance of

MEE cells during palatal fusion. These involve Tak1 functioning upstream of p38Mapk, Smad4 and a novel Smad2 binding partner and a putative chromatin reader Trim33 (Fig. 3-6). While the roles of Tak1 and Trim33 appear fully cooperative with Smad4, our results also reveal a subtle but consistent, and previously unappreciated, non-cooperative role for Smad4 in MEE cells during palatogenesis.

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Chapter 4:

Control elements targeting Tgfb3 expression to the palatal epithelium are located

intergenically and in introns of the upstream Ift43 gene

4-1. Summary

Tgfb3 is strongly and specifically expressed in the epithelial tips of pre-fusion palatal shelves where it plays a critical non-redundant role in palatal fusion in both medial edge epithelial (MEE) cells and in a thin layer of flattened peridermal cells that covers the MEE. It is not known how Tgfb3 expression is regulated in these specific cell types. Using comparative genomics and transgenic reporter assays, we have identified cis-regulatory elements that could control Tgfb3 expression during palatogenesis. Our results show that a 61-kb genomic fragment encompassing the Tgfb3 gene drives remarkably specific reporter expression in the MEE and adjacent periderm. Within this fragment, we identified two small, non-coding, evolutionarily conserved regions in intron 2 of the neighboring Ift43 gene, and a larger region in the intervening sequence between the Ift43 and Tgfb3 genes, each of which could target reporter activity to the tips of pre-fusion/fusing palatal shelves. Identification of the cis-regulatory sequences controlling spatio-temporal Tgfb3 expression in palatal shelves is a key step towards understanding upstream regulation of Tgfb3 expression during palatogenesis and should enable the development of improved tools to investigate palatal epithelial fusion.

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4-2. Introduction

Failure of palatogenesis (palate formation) results in cleft palate, which is one of the most common congenital birth defects in humans. In mice, palatogenesis starts around embryonic day

11.5 (E 11.5) when bilateral outgrowths of the maxillary process called palatal shelves start to grow down vertically on each side of the tongue. Co-ordinated growth of palatal shelves themselves, the tongue and the rest of the oral cavity is followed by rapid palatal shelf elevation

(~ E14) and fusion (~E15) (Bush and Jiang, 2012). Palatal shelves are composed of the neural crest-derived mesenchyme covered by epithelial cells (Bush and Jiang, 2012). Before palatal fusion, the epithelial layer is composed of a basal layer of cuboidal medial edge epithelial (MEE) cells and an apical periderm layer of flattened cells. This periderm layer is shed from the tips of the apposed elevated palatal shelves just before they form contact with each other, allowing adhesion and intercalation of the underlying MEE cells (Yoshida et al., 2012) to form a midline epithelial seam. Epithelial cells in this seam are subsequently lost and the underlying basement membrane degraded resulting in palatal mesenchymal confluence (Gritli-Linde, 2007).

Several studies have demonstrated that signaling triggered by transforming growth factor-

β3 (TGF-β3) plays a critical role in palatal epithelial fusion. Tgfb3 is strongly and specifically expressed in MEE cells (Fitzpatrick et al., 1990; Millan et al., 1991; Pelton et al., 1990), and mice lacking Tgfb3 display 100% penetrant cleft secondary palate (Kaartinen et al., 1995;

Proetzel et al., 1995), which results from defects in TGF-β3-induced palatal MEE differentiation and/or apoptosis (Ahmed et al., 2007; Iwata et al., 2013; Kaartinen et al., 1997; Taya et al.,

1999). Results of a recent study also suggest that TGF-β3 is required for peridermal desquamation (Wu et al., 2013). Mutations in the human TGFB3 have been linked to cleft palate

(Carinci et al., 2007; Lidral et al., 1998), and a recent report described a disease-causing

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mutation in the coding region of TGFB3 in patients showing abnormalities in palate and muscle development(Rienhoff et al., 2013).

A commonly used approach to study complex developmental processes has been to manipulate gene function in mouse models using the Cre-lox system (Rajewsky et al., 1996). In the context of palatogenesis, an epithelium-specific keratin14-Cre (K14-Cre) driver line (Andl et al., 2004) has been frequently used, since it recombines with a very high efficiency in the MEE

(Dudas et al., 2006; Xu et al., 2006). Yet abrogation of the Tgfbr1 gene encoding the TGF-β type

I receptor (Dudas et al., 2006) or Tgfb3 in the palatal epithelium (this study) resulted in a significantly milder palatal phenotype than systemic deletion of the Tgfb3 gene encoding the

TGF-β3 ligand (Kaartinen et al., 1995; Proetzel et al., 1995). Here we show that this phenotypic difference is likely caused by an inability of the K14-Cre driver to recombine in peridermal cells.

To better understand how gene expression is specifically directed in the pre-fusion MEE and overlying peridermal cells, we decided to identify control elements responsible for palate- specific Tgfb3 expression. We surveyed more than 400 kilobases (kb) of mouse genomic DNA sequences on mouse chromosome 12, and identified a 61-kb fragment around the Tgfb3 gene that directs reporter expression specifically in the MEE and adjacent periderm. Using transient transgenic approaches, we identified three smaller cis-regulatory regions: one in the proximal intergenic region and two in intron 2 of the upstream Ift43 gene. These more distal elements may function as ‘shadow’ enhancers assuring robust and reliable control of Tgfb3 expression in the

MEE and adjacent periderm.

4-3. Experimental Procedures

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4-3.1. BACs and BAC recombineering

Mouse BACs RP23-76M13 (=5’BAC) and RP24-299H18 (=3’BAC) were obtained from

Children’s Hospital Oakland Research Institute (http://bacpac.chori.org) (see Fig. 4-2A). Their identity was verified using a standard restriction mapping technique (data not shown).

4-3.2. Insertion of the SA-lacZ-PA cassette into exon1 of the 5’ BAC RP23-76M13 and the 3’

BAC RP24-299H18 (see Fig. 4-2A)

Targeting arms were generated by PCR using BAC RP23-76M13 as a template and the following primers:

Tgfb3-L1: 5’-TCCTAGCTCTACCCAGCACACG-3’

Tgfb3H3Xh-L2:5-’AAGCTTCTCGAGTGTGTGAGCCCAGGAACGAG-3’

Tgfb3XhH3-R1:5’-CTCGAGAAGCTTGCAAAGGGCTCTGGTAGTCCTG-3’

Tgfb3R2: 5’-TGATAGGGGACGTGGGTCATC-3’ pNASSβ (SA-lacZ-PA cassette) was inserted into exon 1 of the BACs RP23-76M13 and RP24-

299H18 using standard BAC recombineering techniques (Warming et al., 2005). Neo 452 (a loxP-Neo-PA-loxP cassette) was added to the generated BAC to enable selection with kanamycin. Integrity of the recombineered BACs was confirmed by PCR after amplification.

4-3.3. Preparation of the 61-kb and 28-kb BACs

The 61-kb BAC: A 128-kb 3’fragment from the recombineered BAC RP24-299H18 was deleted in two steps. First, a targeting vector to replace the large 3’fragment with pGalK was generated by using the primers:

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3’del-F: 5’-

TGACAGATATAGGCAGTGTAAGAACTCGCCATTAGCGGGAGGCGCCATCAGTGCCC

CTTCTGAATTCTACCTGTTGACAATTAATCATCGGCA-3’

3’del-R:

5’-

CTTTTCCCCTTGAGATAAGGCCTCTCATTGAACCTGAAACTTACTTTGATTGGGCTGG

CTT

CAGCACTGTCCTGCTCCTT-3’

After successful recombineering, a targeting vector to delete the pGalK selection marker was generated by PCR using the following primers:

3’del-pGalK-F:

5’-AGAACTCGCCATTAGCGGGAGGCGCCATCAGTGCCCCCTTCTGAATTCTAACAAA

GTCTATACAGTTCCTCACCCTCTGGGAAAAGTAAGTGCTCAAAAC-3’

3’del-pGalK-R:

5’-GTTTTGAGCACTTACTTTTCCCAGAGGGTGAGGAACTGTATAGACTTTGTTAG

AATTCAGAACGGGGGCACTGATGGCGCCTCCCGCTAATGGCGAGTTCT-3’

The targeting vector was deleted as described (Warming et al., 2005).

The 28kb BAC: A 33-kb 5’ fragment was deleted from the 5’end of the 61-kb BAC as outlined above. Primers to generate the targeting vector were:

5’3’del-F:

5’-

TGACCAGGGAGAGGGGCTGTTATGAGGTACTGGGCATCCTGATGGGATGAGAGAAC

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ATTCTCCTGTTGACAATTAATCATCGGCA-3’

5’3’del-R:

5’-GGGCAATGGAGATGTCAAACACGGGCTGCCTAATCTGGAAAGGCATTATTTT

AACTTGTATCAGCACTGTCCTGCTCCTT-3’

The targeting vector to delete the pGalK selection marker was generated by PCR and the following primers:

5’3’del-pGalK-F:

5’-AGGGGCTGTTATGAGGTACTGGGCATCCTGATGGGATGAGAGAACATTCTTA

CAAGTTAAAATAATGCCTTTCCAGATTAGGCAGCCCGTGTTTGACATC3’

5’3’del-pGalK-R:

5’-GATGTCAAACACGGGCTGCCTAATCTGGAAAGGCATTATTTTAACTTGTAAGAA

TGTTCTCTCATCCCATCAGGATGCCCAGTACCTCATAACAGCCCCT-3’

BAC DNAs were purified for microinjections using Nucleobond AX alkaline lysis protocol according to the manufacturer’s instructions (Clontech).

4-3.4. Preparation of smaller reporter constructs

The 2xcHS4-hsp68-lacZ-PA-2xcHS4 vector was generated by replacing a SacII-SacI fragment from the pUbC-SH-Gm-4xcHS plasmid (kindly provided by R. Behringer) with the hsp68-lacZ-

PA cassette. A unique NotI site just upstream of the hsp68 minimal promoter was generated by using the Quikchange-II site-directed mutagenesis kit (Agilent). Regions of interest were PCR- amplified using SuperMix High Fidelity polymerase (Invitrogen) (primer sequences shown in

Table 4-1), and the generated fragments inserted into the NotI site using the In-Fusion HD

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cloning kit (Clontech). Plasmid DNAs were purified using endonuclease-free Maxi-Prep columns (Qiagen) and the purified DNAs were linearized by SalI for microinjection.

4-3.5. Alignment of orthologous sequences and identification of putative binding motifs

Multi-species sequence comparisons around the Tgfb3 gene were performed using the UCSC genome browser (http://genome.ucsc.edu) and VISTA tools for Comparative Genomics

(http://genome.lbl.gov/vista) using the global pair-wise and multiple alignment (LAGAN) program. The threshold used for evolutionary conservation was 70% sequence similarity within

100bp region of DNA sequence. Predicted transcription factor binding sites were identified by using RankVISTA and TRANSFAC matrices.

4-3.6. Generation of transgenic mouse lines and transient transgenic mouse embryos

The transgenic mouse lines and transient transgenics were generated in the Transgenic Animal

Model Core facility at the University of Michigan – Ann Arbor.

4-3.7. Other mouse lines used in this study

We generated epithelium-specific Tgfb3 mutants by crossing mice heterozygous for the floxed

Tgfb3 allele (Tgfb3FXWT) (Doetschman et al., 2012) and carrying the epithelial K14-Cre driver

(Andl et al., 2004) with homozygous floxed Tgfb3 (Tgfb3FXFX) mice. R26R-YFP reporter mice were obtained from the Jackson Laboratories, and generation of Tgfb3-Cre mice has been previously described (Yang et al., 2008).

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4-3.8. X-Gal staining

To detect expression of β-galactosidase encoded by the lacZ reporter gene, embryos were collected, washed and fixed in freshly prepared 4% para-formaldehyde-0.5% glutaraldehyde for

20 minutes, washed 3 x 20 minutes in the detergent wash solution and stained from 4 hours to overnight in X-Gal staining solution as described (Behringer, 2003). The stained samples were examined using a Leica MZ95 dissecting microscope and photographed using an Olympus DP71 camera and DP controller and manager software. Selected samples were processed for paraffin embedding using Histoclear, sectioned, rehydrated and mounted in Immumount (Fisher) or couterstained with eosin or Nuclear Fast Red and mounted in DPX.

4-3.9. Histology and Immunohistochemistry

For paraffin embedding, embryos were harvested and fixed in 4% para-formaldehyde for 24 hours at +4oC, washed, dehydrated and embedded in Leica Histowax. Sections (7 µm) were stained with hematoxylin and eosin using standard protocols. For immunohistochemistry, the paraformaldehyde fixed samples were allowed to sink in sterile 10% sucrose in PBS, then in 7% gelatin/15% sucrose in PBS, oriented and embedded in fresh 7% gelatin/15% sucrose in PBS on ice, then dry ice, and stored at -80oC. Cryosections (10µm) were cut and stored at -80oC. The sections were stained with αSSEA-1 (MC-480 from DSHB) and αGFP (A11122 from Life

Technology) antibodies, which were detected by Alexafluor-594 and Alexafluor-488 secondary antibodies (Invitrogen) respectively. The stained sections were mounted with Vectashield mounting medium containing DAPI (Vector Labs Inc). Sections were viewed using an Olympus

BX51 microscope and documented using an Olympus DP71 digital camera as described above.

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Table 4-1. Primer sequences used for In-Fusion cloning.

Fragment Forward primer Reverse Primer -(6.1-0.8) TTGGCGCCTCCCGCGGCCGCgatgagc GTTTGGATGTTCGCGGCCGCcctttctaagaggcctg ccggcgtcccatctt gttctgg -(6.1-3.7) TTGGCGCCTCCCGCGGCCGCgatgagc GTTTGGATGTTCGCGGCCGCtctctgagaagctggg ccggcgtcccatctt agtctg

-(3.7-0.8) TTGGCGCCTCCCGCGGCCGCttgaatca GTTTGGATGTTCGCGGCCGCcctttctaagaggcctg tttgagaagtgagttt gttctgg

-(13.7-6.1) TTGGCGCCTCCCGCGGCCGCggatcct GTTTGGATGTTCGCGGCCGCgtcgactcaggctgag tctctgtaaagtagac aatt

-(13.7-9.7) TTGGCGCCTCCCGCGGCCGgatccttct GTTTGGATGTTCGCGGCCGCgtgctgcgagccaact ctgtaaagtagac gagcc

-(9.7-6.1) TTGGCGCCTCCCGCGGCCGCcatcagg GTTTGGATGTTCGCGGCCGCgtcgactcaggctgag ttagctggaac aatt

-(7.9-7.6) TTGGCGCCTCCCGCGGCCGCggcaag GTTTGGATGTTCGCGGCCGCcccccctggaaacag ccctgtgtctccct ggtgt

-(7.4-6.6) TTGGCGCCTCCCGCGGCCGCcacacac GTTTGGATGTTCGCGGCCGCaggcactgggatcag acccctgcacaac gc -(13.0-12.5) TTGGCGCCTCCCGCGGCCGgatggagc GTTTGGATGTTCGCGGCCGCggggagcagggttgg cgctgattctga aatcc -(26.9-24.0) TTGGCGCCTCCCGCGGCCGCagacca GTTTGGATGTTCGCGGCCGCggaactaacacttgtc aggtctgcaagt ctg Capital letters indicate the sequences that are homologous to the vector.

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4-4. Results

4-4.1. Palatal peridermal cells are not recombined in a commonly used K14-Cre mouse line

Comparison of the palatal phenotypes of global Tgfb3 knockout mice (Tgfb3-/-) and epithelium-specific Tgfb3 (Tgfb3:K14-Cre) mice revealed that, despite the efficient recombination in the MEE, the germline mutants consistently displayed a more severe phenotype than the tissue-specific mutants (Fig. 4-1, A-I): Tgfb3-/- mice had a complete cleft of the secondary palate (Kaartinen et al., 1995; Proetzel et al., 1995), while Tgfb3:K14-Cre mice had a cleft anteriorly, but superficial or complete fusion in the mid-palate, and an aberrant posterior epithelial bridge. Since the Tgfb3:K14-Cre palatal phenotype was practically identical to that observed in the epithelium-specific TGF-β receptor mutants (both Tgfbr1:K14-Cre and

Tgfbr2:K14-Cre) (Dudas et al., 2006; Xu et al., 2006), we wondered whether this milder palatal phenotype was caused by an inability of the K14-Cre driver line (Andl et al., 2004) to induce recombination in peridermal cells. To address this question we harvested tissues from K14-Cre,

R26R-YFP reporter embryos at E13.5, and assessed the Cre-induced recombination in MEE and peridermal cells (Fig. 4-1, J and K). Our results showed that while the MEE was efficiently recombined, we could not detect reporter expression in the adjacent periderm. In contrast, Tgfb3 was strongly and specifically expressed both in the periderm and underlying MEE as demonstrated by both in situ hybridization, and R26R-lacZ reporter expression in the Tgfb3-

CreKI mouse line (Yang et al., 2008) (Fig. 4-1, L and M).

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Figure 4-1. Milder palatal phenotype of epithelium-specific Tgfb3:K14-Cre mutants than that of Tgfb3 null mutants results from an inability of K14-Cre to recombine in peridermal cells. A-C, control; D-F, Tgfb3-/- mutant; G-I, Tgfb3:K14-Cre (A-I, frontal orientation; all at E18). A, D, G, on the level of the nasal septum (anterior); B, E, H, mid-eye level (middle); C, F, I, on the level of soft palate (posterior). Asterisks in A-C indicate confluent midline mesenchyme, black arrows in D-E point to unfused palatal shelves, black arrows in G-I point to unfused elements of the anterior palate (G), a persistent epithelial seam in the mid-palate (H), and an epithelial bridge in the posterior soft palate (I). J, a frontal palatal section of a K14-Cre:R26R-YFP embryo at E13; double immuno-fluorescence staining to detect YFP-positive recombined cells (green) and an SSEA1-positive subset of non-recombined peridermal cells (red, white arrow). Light blue arrow points to the DAPI-positive nucleus of a peridermal cell that is SSEA-1-negative and has not been recombined by K14-Cre. K, a frontal palatal section of a X-Gal-stained K14-Cre:R26R- lacZ embryo at E13, counterstaining with eosin. Black arrows point to apical peridermal cells that were not recombined with K14-Cre. L, in situ hybridization for Tgfb3 at E14 (palatal frontal section). Black arrow points to a positively staining flattened cell with peridermal appearance. M, a frontal palatal section of X-Gal-stained Tgfb3-Cre:R26R-lacZ embryo, counterstained with eosin. Black arrow points to an X-Gal-positive flattened cell with peridermal appearance. PS, palatal shelf; NS, nasal septum. Scale bars in A-I, 200µm; J-M, 50µm.

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4-4.2. Survey of the Tgfb3 cis-regulatory function using recombinant reporter BACs

Since Tgfb3 is strongly and specifically expressed in peridermal and MEE cells, we reasoned that identification of cis-regulatory elements controlling palate-specific Tgfb3 expression would be invaluable for development of new improved genetic tools to examine palatal epithelial fusion in vivo. The Tgfb3 gene, composed of 7 evolutionarily conserved exons, is located on mouse chromosome 12 between the Ift43 (intraflagellar transporter 43) and Ttl5

(tubulin tyrosine ligase-like 5) genes, which both lie in the opposite orientation to the Tgfb3 gene

(Fig. 4-2A). The intergenic flanking sequences are remarkably short (3-3.5kb) but the neighboring genes are not expressed in pre-fusion palatal shelves (Fig. 4-4Ab).

To assess large regions upstream and downstream of the Tgfb3 gene for regulatory elements, we obtained two overlapping BACs. The 5’ BAC (RP23-76M13) contained a 289-kb region from -263kb to +26kb (defining Tgfb3 transcriptional start site (TSS) as 0) which included the Tgfb3, Ift43 and Gpatch2l genes (Fig. 4-2A). The 3’ BAC (RP24-299H18) contained the 189-kb region from -35kb to +154kb which included the Tgfb3 gene and the Ttll5

(variant 4) gene (Fig. 4-2A). The sequences of the two BACs overlapped by 61kb, which includes all the Tgfb3 exons and some of those of the neighboring genes.

To prepare reporter constructs, we inserted an SA-lacZ-pA cassette into Tgfb3 exon 1 of each

BAC using standard recombineering techniques (Warming et al., 2005). These recombinant lacZ reporter BACs were used to generate transgenic mice and β-galactosidase activity assessed at

E14.0 using X-Gal staining as Tgfb3 is usually strongly expressed in palatal shelf MEE and nasal septal epithelium around this stage. Both 5’ and 3’ lacZ-BACs were able to target reporter activity correctly to the palatal midline and nasal septal tissues (Fig. 4-2, B and F).

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Figure 4-2: Recombinant reporter BACs used to detect Tgfb3 regulatory regions. A, schematic representation of Tgfb3-containing lacZ BAC clones. Exons of the Tgfb3 gene are shown as red vertical bars and exons of Ttll5, Ift43 and Gpatch21 genes are shown as green, blue and purple vertical bars, respectively. Horizontal black arrows show the direction of transcription of each gene. B-E, 5’ lacZ BAC transgenic embryos showing β-gal reporter activity (blue staining) in tips of palatal shelves (B, black arrows), nasal septum (B, asterisk, at anterior end of palate), blood vessels (C, green arrow), mammary placodes (C, red arrows) and in whisker follicles (D, E, blue arrows). F-I, 3’ lacZ BAC transgenic embryos showing β-gal reporter activity (blue staining) in tips of palatal shelves (F, black arrows), nasal septum (F, asterisk), blood vessels (F, G, green arrows). B, F, roof of mouth at E14, inferior view; C, G, torso at E14, right lateral view, D, H, head (mandible removed), left lateral view; E, I, head (mandible removed), frontal view. J-N, Reporter activity in 3’ lacZ-BAC transgenic embryos between embryonic days 10.5 and 16.0. Black arrows point to expression in tips of pre-fusion palatal shelves (K, L) and midline seam (N); green arrows point to positively staining blood vessels (K,

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L, M, N); asterisk (in M) marks the nasal septum PS, palatal shelf. B, F, K-N, anterior, top; posterior, bottom. Scale bars in K, 450µm; B, F, J, L, M, N, 500µm; C, D, E, G, H, I, 1mm.

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The 5’ lacZ-BAC transgenic embryos were also stained in mammary placodes, whisker follicles, nostrils and vasculature (Fig. 4-2, C-E), while the 3’lacZ-BAC embryos showed additional staining principally in vasculature (Fig. 4-2G, and Fig. 4-3, which illustrates the positions of all DNA fragments tested for enhancer activity in this study and summarizes expression data). Stable transgenic mouse lines carrying 3’ lacZ-BAC did not show detectable reporter activity until E12.0-E12.5, when staining was seen first in blood vessels and soon afterwards in the tips of the posterior palatal shelves (Fig. 4-2, K-L). At E14.5 strong staining occurred along the entire A-P axis of the palatal shelf tips, and continued during and after palatal epithelial fusion when it could still be detected in the degrading midline seam and in vasculature at E16.0 (Fig. 4-2, M and N).

As the 5’ and 3’ BAC sequences overlapped, and both the 5’ and 3’ lacZ-BAC reporters drove expression in the palatal shelf tips, we hypothesized that sequences in the region common to each BAC may be responsible. We tested this by making a reporter lacZ-BAC containing only this sequence, from -35kb to +26kb (Fig. 4-3A, 4-4Aa). This 61-kb fragment of mouse genomic

DNA consistently drove highly specific reporter expression in the MEE and the adjacent periderm, and in peridermal cells covering the nasal septum where anterior secondary palatal fusion occurs (Fig. 4-4B, F, G, H). The only other tissue showing detectable though weak X-Gal staining was the lens (Fig. 4-4, D and E).

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Figure 4-3. Enhancer screening across the Tgfb3 region. A, Schematic presentation of the regions examined to locate cis-regulatory sequences directing reporter activity to the MEE/periderm cells. (a) 417-kb of genomic DNA (black line) including the Tgfb3 gene (red boxes represent exons); colored lines beneath correspond to the positions of BAC sequences

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used for expression regulation analysis (see B, first four columns). (b) The 61-kb region in common between the 5’ and 3’ BACs (brown line); graph above shows evolutionary sequence conservation among placental mammals (UCSC genome browser) along this sequence; red boxes represent the Tgfb3 exons; colored lines beneath correspond to the positions of regions used for regional expression regulation analysis (see B; fourth column onwards); orange pentagons illustrate the ECRs I-VII (see main text). B, a table summarizing findings of reporter activity driven by the sequences in the regions indicated by colored bars in A in various tissues of transgenic embryos at E14. Data entries show the number of embryos displaying positive reporter activity in selected tissues (rows) over the total number of lacZ-positive embryos for each sequence (identified by color bar in each column; dark yellow highlight, 50% or more staining; pale yellow highlight, >0%, <50% staining.

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Figure 4-4. A 61-kb genomic region including theTgfb3 gene targets reporter activity to the MEE and adjacent periderm. A, Schematic representation of the 61-kb region (see main text) includes (a) the Tgfb3 gene (exons in red); exons 1-3 of the Ift43 gene (blue boxes) and exon 16 of the Ttll5 gene (green box). That Tgfb3 but neither of its neighbors is actively expressed at E14.5 is shown in (b, RNA-seq profile in mouse palate at E14.5 -FaceBase Enhancer Project; A Visel). Different types of sequence analysis are suggestive of possible enhancer sites: (c) p300 Chip-seq profile in mouse palate at E14.5 (ref: FaceBase Enhancer Project; A Visel), (d) evolutionary sequence conservation among selected vertebrate species in this sequence (UCSC genome browser) and (e) evolutionary sequence conservation among placental mammals (UCSC genome browser). B-E, 61-kb BAC-lacZ embryos showing β-gal activity (blue staining) in tips of palatal shelves (B, black arrows), nasal septum (B, asterisk), and lens (D, E, red arrows). B, mouth roof at E14, inferior view, anterior on the top; C, torso at E14, right lateral view; D, head (mandible removed), left lateral view; E, head (mandible removed), frontal view. F-H, Frontal sections of an X-Gal stained 61-kb lacZ-BAC embryo at the level of nasal septum (indicated by the red line in B). F, X-Gal staining (black arrows) at the tips of the pre-fusion palatal shelves (PS) and in the nasal septum (NS) is present in cells of both the basal MEE layer (red arrows in G) and the overlying periderm layer (black arrows in G), and periderm in the nasal septum (black arrow in H). Scale bars in B, 500µm; C, D, E, 1mm; F, 200µm; G, H, 50µm.

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4-4.3. Noncoding evolutionarily conserved sequences within the Tgfb3 gene are not responsible for the MEE-specific gene expression

We analyzed the 61-kb overlapping region using the UCSC Genome Browser

(http://genome.ucsc.edu) (Fig. 4-4Ac-e) to identify non-coding evolutionarily conserved regions

(ECRs), which are likely to include tissue-specific enhancers and found four: ECR-I (2kb upstream of TSS), ECR-II and ECR-III (in Tgfb3 intron 1) and ECR-IV (in Tgfb3 intron 3) (Fig.

4-5). As these are all highly conserved in placental mammals, which develop a complete secondary palate, but not in avians (or fish), which do not express Tgfb3 in tips of palatal shelves and do not develop the fused secondary palate (Sun et al., 1998), they were good candidates to regulate palate-specific expression. To test this, the ECRs (I-IV) were PCR-amplified and subcloned upstream of the minimal hsp68 promoter and lacZ-PA reporter, and the resulting

ECR-(I-IV)–hsp68-lacZ-PA cassette cloned between concatamerized pairs of genomic insulators

(cHS4), which have been shown to reduce positional effects of transgenes and alleviate promoter interference(Griswold et al., 2011);Yahata, 2007 #3977}(Fig. 4-5). Transient transgenic embryos were generated and analyzed for reporter activity at E14.0. While the ECRs were consistently able to target the reporter activity to several tissues (teeth, whisker follicles, nostrils and forebrain), no staining was seen in the MEE (n=15) (Fig. 4-5). We also modified the 61-kb lacZ-

BAC by deleting sequences from -35kb to -3kb. This 28-kb BAC was also unable to direct lacZ reporter expression to the MEE (Fig. 4-3). These data suggest that the sequences from -3.0kb to

+26kb, including the entire Tgfb3 gene and ECRs I-IV, are not responsible for the

MEE/periderm-specific gene expression in mouse embryos during palatogenesis.

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4-4.4. Cis-regulatory elements directing gene expression in the MEE are located in intron 2 of the upstream Ift43 gene

In addition to the noncoding ECRs I-IV, the 61-kb region from -35kb to +26kb contains three additional highly conserved regions in intron 2 of the Ift43 gene: ECR-V at position –(7.9 -

7.6)kb, ECR-VI at -(13.0 - 12.5)kb and ECR-VII at –(26.9 – 24.0)kb. To assess these regions for the possible presence of MEE/periderm-specific cis-regulatory elements, we first subcloned the

ECRs -V and -VI into the 2xcHS4-hsp68-lacZ-2xcHS4 vector as a single 7.6-kb fragment (Fig.

4-5A) and ECR-VII as a 2.9-kb fragment (Fig. 4-5A). Analysis of X-Gal-stained transient transgenic embryos at E14 revealed that the region surrounding ECR-VII targeted the reporter activity to vascular and skeletal structures, but did not direct reporter activity in the MEE (Fig. 4-

3, and data not shown). In contrast, the 7.6-kb region containing both ECRs -V and -VI was able to drive expression not only in the MEE/periderm with high efficiency (7/14) (Fig. 4-3B and Fig.

4-6C, V, X), but also in the vasculature (including palatal vessels), nostrils and whisker follicles

(Fig. 4-6C-F). Within this region, ECR-V is conserved only in mammals but ECR-VI is conserved in both mammals and avians suggesting that ECR-V would be more likely to contain palate-specific control elements. Indeed, this was the case, since the sequences between -9.6kb and -6.1kb including ECR-V targeted reporter activity to the MEE/periderm (Fig. 4-3 and Fig. 4-

6H, Y, Z), while ECR-VI and surrounding sequences (from -13.7kb to -9.7kb) did not (Fig. 4-3 and data not shown). To narrow down the regions within -9.7kb and -6.1kb that contained putative cis-regulatory modules, we next examined a 0.3-kb fragment that encompassed the highly conserved ECR-V (from -7.9kb to -7.6kb), and an adjacent conserved 0.8-kb region from

-7.4kb to -6.6kb (Fig. 4-6A). Each region drove reporter expression in the tips of palatal shelves but relatively weakly (Fig. 4-3 and Fig. 4-6M, R) and with far less specifically than the larger (-

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13.7 - -6.1kb) fragment. These results suggest that the 3.5-kb region in Ift43 intron 2 contains two or more cis-regulatory elements independently able to direct the reporter activity in the MEE and adjacent periderm, but they are needed in combination to drive expression efficiently.

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Figure 4-5. Evolutionarily conserved regions within the Tgfb3 gene do not direct reporter expression in palatal shelves. A, Schematic representation (a) of the Tgfb3 gene (red boxes depict Tgfb3 exons 1-7; blue box depicts Ift43 exon 1; black arrows show the TSSs for Tgfb3 and Ift43) aligned with graphs of evolutionary sequence conservation among selected vertebrate species (b, UCSC genome browser), among placental mammals (c, UCSC genome browser) and RNA-seq profile in mouse palate at E14.5 (d, FaceBase Enhancer Project; A Visel)). Grey boxes indicate non-coding, evolutionarily conserved regions (ECRs I-IV). Purple line indicates the region present in the 29-kb lacZ-BAC. B, Schematic presentation of the reporter construct used to generate transgenic embryos shown in C-F. C-F, ECR(1-IV)-hsp68-lacZ embryos at E14 showing β-gal activity (blue staining) in molars (C, black arrows), ribs and lower spine (D, red arrows) and forebrain (F, green arrow). C, mouth roof at E14, inferior view, anterior on the top; D, torso at E14, right lateral view, E, head (mandible removed), frontal view; F, head (mandible removed), left lateral view. Scale bars in C, 700 µm; D, E, F, 1mm.

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Figure 4-6. Cis-regulatory elements targeting reporter activity to the MEE and adjacent periderm are located in intron 2 of the upstream Ift43 gene. A, a 28-kb sub-region of the 61- kb genomic fragment (a, see Fig. 4 and the main text) that includes Tgfb3 exon 1 (red box) and exons 1 and 2 of the Ift43 gene (blue boxes), (b) evolutionary sequence conservation among selected vertebrate species (UCSC genome browser), and (c) among placental mammals (UCSC genome browser), and (d) RNA-seq profile in mouse palate at E14.5 (FaceBase Enhancer Project; A Visel). Vertical rectangles depict the non-coding ECRs -V, -VI and -VII. Colored bars below (d) correspond to DNA fragments (see also Figure 3) examined by transient transgenic reporter assay in constructs shown schematically above images of the stained embryos generated (B-Y). C-F, Transgenic reporter embryos carrying a 7.6-kb DNA fragment from -13.7 to -6.1kb (green bar below (c) and green rectangle in construct schematic) showing β-gal activity (blue staining) in tips of palatal shelves (C, black arrows), blood vessels (C, D, green arrows) and nostrils (F, red arrow). H-K, Transgenic reporter embryos carrying a 3.6-kb DNA fragment from -9.7 to -6.1kb (-blue-green bar below (c) and blue-green rectangle in construct schematic) showing β-gal activity(blue staining) in tips of palatal shelves (H, black arrows), lens (J, blue arrow) and nostrils (K, red arrow). M-P, Transgenic reporter embryos carrying a 0.3-kb DNA fragment (ECR-V) from -7.9 to -7.6kb (light blue bar below (c) and light blue rectangle in construct schematic) showing β-gal activity (blue staining) in tips of palatal shelves (M, black arrows), apical ectoderm (N-P) and nostrils (P, red arrow). R-U, Transgenic reporter embryos carrying a 0.8-kb DNA fragment from -7.4 to -6.6kb (turquoise bar below (c) and rectangle in construct schematic) showing β-gal activity (blue staining) in tips of posterior palatal shelves (R, black arrows). V-X, Frontal sections of the X-Gal-stained 7.6-kb fragment transgenic embryo shown in C at the level of the posterior palate (indicated by the red line in C). Staining at the tips of palatal shelves (black arrows in V) is in both MEE cells (red arrow in X) and periderm cells (black arrow in X). Y-Z, Frontal sections of the X-Gal-stained 3.6-kb fragment transgenic embryo shown in H at the level of the posterior palate (indicated by the red line in H). Staining at the tips of palatal shelves (black arrows in Y) is in both in MEE cells (red arrow in Z) and periderm cells (black arrow in Z). Scale bars in C, H, M, R, 500µm; D, I, N, S, E, F, J, K, O, P, T, U, 1mm; V,Y, 100 µm; X, Z, 50µm.

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4-4.5. An additional cis-regulatory region is located in a 5.3-kb fragment immediately upstream of Tgfb3 exon 1

Since the overall conservation of the intergenic region between the Ift43 and Tgfb3 genes is relatively high, we examined whether this region could also contribute to MEE/periderm- specific expression. First we cloned the 9.8-kb region from -6.1kb to +3.7kb (i.e., Ift43 intron 1,

Ift43 exon 1, intervening sequences between the Ift43 and Tgfb3 genes and Tgfb3 exon 1) between concatamerized pairs of cHS4 insulators, and inserted a lacZ-PA cassette in frame into the Tgfb3 exon 1 (Fig. 4-7B). This construct, driven by the endogenous Tgfb3 promoter, directed reporter activity specifically in the palatal midline region in transient transgenic embryos, although with a relatively low frequency (2/5) (Figs. 4-3B and 4-7C). To further define the important region within this 9.8kb fragment, we subcloned the 5.3-kb region from -6.1kb to -

0.8kb into the 2xcHS4-hsp68-lacZ-2xcHS4 vector, as it lacks the endogenous Tgfb3 promoter

(Fig. 4-7G). Two of the three resulting transgenic embryos showed reporter activity in the palatal midline (Fig. 4-7H) though also in several other tissues (Fig. 4-3 and 4-7I-K), suggesting that the shorter sequence lacked elements necessary for highly regionally specific regulation. Intron 1 of

Ift43 (from -6.1 to -3.7kb) alone, or the intervening sequence between Ift43 and Tgfb3 (from -3.7 to -0.8kb), did not direct β–galactosidase activity in the tips of palatal shelves (n=5 in each case)

(Fig. 4-3B and data not shown). These data imply that a fragment from Ift43 intron 1 to Tgfb3 exon 1 contains a putative proximal MEE/periderm enhancer, which is dependent on DNA sequences separately located in smaller fragments.

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Fig. 4-7. A putative proximal enhancer directing palatal expression lies in a 5.3-kb region upstream of the Tgfb3 gene. A, Schematic representation (a) of a 28-kb region upstream of Tgfb3 exon 1 (red box) including Ift43 exons 1 and 2 (blue boxes) aligned with (b) evolutionary conservation among selected vertebrate species (ucsc genome browser), (c) evolutionary conservation among the placental mammals (ucsc genome browser), (d) RNA-seq profile in mouse palate at E14.5. Colored bars below (d) correspond to DNA fragments examined using transient transgenic reporter assays (B-N). B, G, Schematic presentations of reporter constructs used to generate transgenic embryos (colored rectangles correspond to colored bars shown above (a)). C-F, Transgenic reporter embryo carrying the 9.8-kb DNA fragment from -6.1 to +3.7kb (magenta bar above (a) and magenta rectangle in B) showing β-gal activity (blue staining) in tips of palatal shelves (C, black arrows) and in the nasal septum (asterisk in C). No staining was seen in the torso (D) or head (E, F). H-K, Transgenic reporter embryo carrying the 5.3-kb DNA fragment from -6,1 to -0.8kb (dark purple bar above (a) and rectangle in G) showing β-gal activity (blue staining) in tips of palatal shelves (H, black arrows), in nasal septum (asterisk in H), in the ectoderm (red asterisks in H, I) and in skeletal structures (I, blue arrows) and olfactory bulbs (J, red arrows). L-N, Frontal sections of the X-Gal-stained 5.3-kb transgenic embryo shown in H at the level of the nasal septum (indicated by the red line in H). Staining can be seen at the tips of palatal shelves (black arrows in L, M) and in periderm cells of the nasal septum (black arrow in N). Scale bars in C, H, 500µm; D, I, E, F, J, K, 1mm; L, 200µm; M, N, 50µm.

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4-5. Discussion

Conditional Cre drivers are an invaluable tool for investigating the roles and timing of gene expression in processes involving several cell types such as palatogenesis. Key to this is knowledge of their recombination patterns and efficiency. Here we have shown that K14-Cre recombines efficiently in palatal medial edge epithelium (MEE) but it is not expressed in the overlying palatal periderm. This could explain the phenotypic differences between the germline

Tgfb3 mutants (in which no Tgfb3 is expressed by MEE or periderm, periderm is inadequately shed and a complete cleft of the secondary palate occurs) and the epithelium-specific Tgfb3:K14-

Cre mutants (in which genotypically normal periderm itself may be providing sufficient TGF-β3 signaling for some shedding, and thus a milder phenotype occurs). This proposed role for peridermal Tgfb3 expression, and known peridermal responsiveness to TGF-β3-triggered signaling(Wu et al., 2013) combined with an inability of K14-Cre to recombine in peridermal cells could also explain why periderm behaves normally in Tgfbr2:K14-Cre mutants (Iwata et al., 2013). In contrast with these TGF-β3 signaling, K14-Cre conditional knock-outs, epithelium- specific β-catenin mutants (Ctnn1b:K14-Cre) lose Tgfb3 expression in tips of palatal shelves but still develop total cleft of the secondary palate (He et al., 2011), raising the intriguing possibility that canonical Wnt signaling is specifically required in MEE for Tgfb3 expression to occur in both the MEE and adjacent periderm. To test these and related hypotheses other conditional Cre drivers are required: to recombine only in palatal periderm, and to recombine in both MEE and the overlying periderm. It is not clear whether all other Cre-drivers currently used to delete genes in the palatal epithelium recombine in periderm as well, and many have additional limitations:

Pitx2-Cre recombines predominantly in the posterior palatal epithelium (Xiong et al., 2009); recombination in the Foxg1-Cre line is highly background-dependent (Hebert and McConnell,

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2000); and, as Tgfb3 itself is expressed and required in several other tissues besides MEE and adjacent periderm during early embryogenesis, the Tgfb3-Cre knock-in line is of very limited use in studies of palatal epithelial fusion (Yang et al., 2008). As expression of Tgfb3 in MEE and in periderm is so crucial to normal palatogenesis, occurs in precisely the regions where we would like to regulate the expression of other genes genetically, and its regulation poorly understood, we set out to identify the enhancer sequences responsible for this highly specific expression of

Tgfb3. Using BAC deletion analysis we were able to identify a 61-kb region around the Tgfb3 gene that could drive lacZ reporter expression specifically in the MEE and adjacent periderm.

Expression of this reporter was much more specific to the MEE/periderm than that of the endogenous Tgfb3 gene, and as this 61-kb region did not drive detectable expression before

E12.5 it is a good candidate region for the development of novel palatal epithelium/periderm- specific Cre-driver lines.

In order to use enhancer sequence information to learn more about molecular regulation of Tgfb3 expression we needed to identify the important sequences more precisely.

Palatogenesis is an evolutionarily conserved developmental process in amniote animals (Bush and Jiang, 2012). Mammals and reptiles have a fused secondary palate, and although avians develop a beak and have a naturally cleft palate (Ferguson, 1988) fusion can be induced by exposing the appropriate stage avian palatal shelves (which do not express endogenous cTgfb3) to human recombinant TGF-β3 (Sun et al., 1998). Although it was therefore likely that enhancers directing palatal expression would be amongst non-coding highly conserved sequences amongst mammals, these are very numerous, and those that lay within the Tgfb3 gene

(ECRI-IV) turned out not to be palatal enhancers. The FaceBase project (www.facebase.org -A.

Visel) to identify craniofacial transcriptional enhancers using ChIP-Seq (IP using anti-p300)

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recently released a dataset obtained on E14 whole palates but this was not helpful for our specific project; within the 61-kb region only a region around Tgfb3 exon 1 was flagged as being a putative enhancer; we could demonstrate only a putative vascular enhancer in the area of ECR-

VII (Fig. 4-3, Fig. 4-4 and data not shown) where the anti-p300 binding was above the background level in ECR-VII (Fig. 4-4).

By directing our analysis outside the Tgfb3 gene within the 61kb fragment, we were able to identify a distal 3.5-kb region in Ift43 intron 2 and a proximal 5.3-kb region encompassing

Ift43 intron 1 and most of the intergenic sequence between the Ift43 and Tgfb3 genes able to target the reporter activity to the MEE and adjacent periderm. However, these smaller regions directed less specific and weaker reporter activity than the 61kb fragment. While we were able to break the distal 3.5-kb region down further into two smaller modules, which again showed further reduced activity, our attempts to narrow down the 5.3-kb proximal region into even shorter sequences were not successful, suggesting that palate-specific reporter activity seen in the larger region was dependent on two or more regulatory elements separately located in the smaller fragments. Although the same approach has yielded relatively short enhancers that drive very precise and strong expression in other cases (Chandler et al., 2007; Dodou et al., 2004), it is established that not all physically concise and robust expression is regulated in such a simple manner(Evans et al., 2012). Genetic regulatory network studies in Drosophila first introduced the concept of ‘shadow’ enhancers (Lagha et al., 2012). Perry et al reported that, in addition to the proximal primary enhancer located just upstream of the promoter, the snail gene is regulated by a distal enhancer located within the neighboring locus(Perry et al., 2010), which they suggested be defined as a ‘shadow’ enhancer. Subsequent studies have suggested that secondary enhancers are needed to obtain sufficient phenotypic robustness to drive tightly controlled

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expression of important developmental genes (Frankel et al., 2010). Our findings of putative proximal (primary) enhancer(s) and two (or more) distal enhancers in the neighboring upstream gene that work precisely but only weakly in isolation are reminiscent of this mechanism. A ‘lack of simplicity’ may also extend to the organization of enhancers for other tissues and repressive elements controlling Tgfb3 expression as we noticed that, unlike the 61-kb region which directed reporter activity specifically in the MEE/periderm, many of the smaller domains around the

ECR-V also targeted the reporter activity to the vasculature including the palatal vessels. Similar vascular patterns were seen in embryos carrying either ECR-VI or ECR-VII (which were unable to direct expression in the MEE/periderm) suggesting that all three ECRs located in intron 2 of

Ift43 possess putative redundant vascular enhancer activities.

Very little is known about the molecular mechanisms regulating Tgfb3 expression in the epithelial tips of pre-fusion palatal shelves. Venza et al recently reported that in Foxe1 mutant embryos Tgfb3 expression is dramatically reduced in the palatal epithelium, and that Tgfb3 is a direct target of FoxE1 via FoxE1 binding sites in the Tgfb3 promoter region (Venza et al., 2011).

As outlined above, He et al reported that epithelium-specific mouse mutants lacking the gene encoding β-catenin also show a dramatic reduction inTgfb3 expression in the palatal epithelium suggesting that canonical Wnt signaling is involved in Tgfb3 regulation (He et al., 2011).

Whether these identified transcriptional regulators function purely by contributing to the core promoter activity or by also regulating Tgfb3 expression via distal enhancers is not yet known.

Nevertheless, even the smallest cis-regulatory region identified in this study (i.e., the 300-bp

ECR-V located in the Ift43 intron 2) contained three evolutionarily conserved TCF/LEF consensus binding sites and two FoxE1 binding sites (data not shown) implying that these factors may have the capacity to regulate Tgfb3 in palatal shelf tissues by binding directly to the putative

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enhancer elements. Thus our results are consistent with existing molecular regulation data, and suggest a model in which the MEE/periderm-specific Tgfb3 expression is achieved via a complex regulatory landscape composed of a putative proximal (primary) enhancer(s) and two

(or more) distal shadow enhancers i.e. some that lie in the neighboring upstream gene.

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Chapter 5:

Conclusions

5.1. Summary of findings

Previous to work presented in this thesis, Smad-dependent TGF-β signaling within the palatal mesenchyme and epithelium was thought to be the key contributor to forming a functional palate. However, many of the studies that exist have been focused on conditional removal of Smad-dependent signaling components. Here, we show that Smad-independent signaling components Tak1 and Trim33 along with Smad4 play an under appreciated role in palatogenesis.

5.2. Tak1 signaling in the craniofacial neural crest

Our data demonstrate that Tak1 plays a larger role in craniofacial ectomesenchyme than just TGF-β and Bmp-induced activation of p38 Mapk (Smad-independent). Tak1 is responsible for agonist induced c-terminal and linker phosphorylation of Smad2 (Smad-dependent), which is needed for full transcriptional activation. In the absence of Tak1 in the ectomesenchyme, palatal shelves fail to elevate and fuse. Previous to this study, the role of Tak1 in the mesenchyme was unknown; surprisingly Tak1 plays a role in both the Smad-independent and Smad-dependent

TGF-β signaling pathway (Figure 5-2).

Tak1 phosphorylates the c-terminus of Smad2 in the mesenchyme during palatogenesis and when Tak1 is absent, Smad2 is not fully activated. This leads to a reduction of Smad2 in the

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nucleus and therefor a reduction of Smad2 linker region phosphorylation. It has been shown that the linker region phosphorylation at Thr-220 is phosphorylated by CDKs and Mapks (JNK/ERK) phosphorylates Ser-250. When we inhibited CDKs in primary palatal mesenchymal cells, we saw that phosphorylation at both Thr-220 and Ser-250 was reduced. The next step would be to study the exact mechanism of phosphorylation by CDKs and the exact role Tak1 plays on the phosphorylation of the Smad2 linker region in the nucleus. In addition, it would be good to follow up on the “action turnover switch model” where the linker region phosphorylation is needed for full R-Smad activation and cessation of activation, which has never been examined in-vivo. In this model nuclear CDKs phosphorylate Thr-220 in Smad2 and create docking sites for transcriptional co-regulators, which then allows phosphorylation of Ser-250 in Smad2, which ultimately provides a docking site for ubiquitin ligases, which target Smad2 for proteasome- mediated degradation. How phosphorylation of Smad2 mediated by Tak1 provides docking sites for co-regulators and ubiquitin ligases and ultimately turns on and off Smad2 activity would add additional information on the role of the linker region phosphorylation in the larger signaling scheme.

The upstream components that lead to Tak1 activation during TGF-β signaling are not well known. Although, Tak1 is activated by TGF-β2 stimulation, the role of TGFβRI kinase activity on the activation of Tak1 has not been clearly examined. Additionally, there is a clear overlap between TGF-β-induced transcriptional responses dependent on both TGFβRI and Tak1 kinase activities. Our studies tested the role of TGFβRI protein lacking kinase activity with the use of the chemical inhibitor. Exactly how TGFβRI kinase activities or the presence of TgfbrI work to regulate downstream signaling events is an area for future research.

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5.3. Tak1, Trim33 and Smad4 signaling in the pre-fusion palatal epithelium

Tak1 is expressed in the mesenchyme and in the epithelium of the developing palate. We show that when Tak1 alone is removed from the palatal epithelium, there is no resulting palatal phenotype. Interestingly, we found that if Smad4 is removed from the palatal epithelium, there is a subtle, but apparent defect in palatogenesis, which was not addressed in previous studies (Xu et al., 2006). However, if we remove Tak1 and Smad4 together, there is a resulting phenotype, which is more severe than removal of Smad4 alone. We found that this defect resulted in a decrease in TGF-β3 signaling.

It is possible that phosphorylated Smad2 can partner with other transcriptional regulators to regulate target gene expression. We have shown that specific removal of Trim33 alone in the palatal epithelium does not have a phenotype. However, when Trim33 and Smad4 are removed there is persistence of the midline epithelial seam.

Ultimately, Tak1, Trim33 and Smad4 play cooperative roles in MES removal and lead to a complete palate. We found that although the three genes play redundant roles, when two genes are removed (Trim33 and Smad4) along with an inhibitor against the third gene (Tak1), there is a more severe cleft of the secondary palate. (Figure 5-3)

Previously to this study, there has not been any research done on the mechanism by which p38 is activated within the palatal epithelium. We found Tak1 is upstream of p38 and that Tak1 and Smad4 work together to form a complete palate. We also found that when Tak1 and Smad4 are removed it does not result in the same phenotype as the Tgfb3 global or K14-Cre conditional knockouts. This suggests that there are likely more genes involved in the removal of the midline epithelial seam. In the future, it is possible that TGF-β3 signaling along with another

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pathway may work together to form the palate. What these molecules are is something that will need to be examined.

It is known that when Smad2 is activated through TGF-β3 dependent activation, it binds

Smad4 and accumulates in the nucleus leading to target gene regulation. Although, there is advanced knowledge in signaling pathways, little is known about epigenetic alterations that allow the chromatin state to be coordinated with gene expression. Trim33 has been shown to be a histone-binding protein which complexes with Smad2 forming a Trim33-Smad2 chromatin reading complex (Xi et al., 2011). This reading complex recognizes and binds repressive marks on the histone tail, and increases acetylation, which allows Smad4 access to regulate target genes. However, if Trim33 is playing this role in the palatal epithelium is unknown. We have found in this study that Trim33 is needed along with Smad4 for p21 expression leading to complete removal of the midline epithelial seam. Future studies could help to elucidate the exact mechanisms by which Trim33 plays a role in the nucleus leading to target gene expression resulting in removal of the midline epithelial seam.

5.4. Tgfb3 expression in pre-fusion palatal epithelium

How does Tgfb3 get specifically expressed within the MES? We know from previous studies that when B-catenin or Foxe1 is removed from the palatal epithelium it leads to a complete cleft of the secondary palate and a reduction in Tgfb3 expression (He et al., 2011;

Venza et al., 2011). This study has shown for the first time the presence of multiple cis- regulatory elements that drive specific expression of Tgfb3 in the palatal epithelium during palatogenesis (Figure 5-3).

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Not much has been known about the upstream regulation of Tgfb3 expression. Amongst the cis-regulatory elements that we have found, we looked closely for the presence of downstream transcription factors that would bind and drive expression of Tgfb3, LEF1 and FOX transcription factor binding sites (Figure 5-1). A future experiment that could be done would be to use ChIP against the LEF1 and FOX sites and examine where they bound on the Tgfb3 cis- regulatory elements that we have identified. We could then use these areas of binding to determine if they can drive expression and ultimately teach us about the upstream regulation of

Tgfb3.

Finally, the next step in this research would be to make a transgenic mouse line using the cis-regulatory elements that we have found in this study. More specifically, we could use the

61kb fragment, which provided the most specific reporter activity. The mouse line would be useful to remove genes both in the palatal epithelium and periderm. We could then study the roles of genes in both the epithelium and overlying periderm during the exact times of adhesion and fusion.

5-5. TGF-β3 signaling during palatogenesis and health significance

The long-term goal of these studies is to understand the signaling mechanisms that are needed for proper palatogenesis. We can then prevent clefting in patients and remove the long-term nutritional and societal issues for these individuals.

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Figure 5-1. LEF1 and FOX sequence alignment to identified cis-regulatory elements of Tgfb3. A. Regions of the Tgfb3 gene are shown with conservation between mouse and human. Above each area of conservation there are green lines that indicate the presence of either a LEF1 or FOX binding site. B. A larger detailed view of the blue-boxed region in A, where the upper sequence refers to mouse and the lower sequence refers to human. Pink highlighted regions indicate the presence of a LEF1 site and the blue highlighted regions indicate the presence of a FOX binding site. If both LEF1 and FOX binding sites are present, it is indicated in purple. C. A larger detailed view of the red-boxed region in A. The presence of one LEF1 site is indicated. D. A larger detailed view of the green-boxed region in A. The presence of one LEF1 site is indicated.

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Figure 5-2. Schematic representation of mesenchymal Smad2 activation via Tak1.

Upon TGF-β activation, Smad2 C-terminal phosphorylation is increased in the presence of Tak1. Cdk-dependent phosphorylation of Threonine-220 occurs in the nucleus along with Jnk/ERK dependent phosphorylation of Serine-250.

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Figure 5-3. Schematic representation of Tak1, Smad4 and Trim33 redundant functions during palatal epithelial fusion.

Tak1 and Smad2 are activated upon TGF-β3 activation. Tak1 activates p38 where is performs a redundant role in leading to medial edge epithelium (MEE) apoptosis. Activated Smad2 binds to the common co-mediator, Smad4 where Smad2/Smad4 accumulate in the nucleus and play a redundant role in MEE apoptosis. Some nuclear activated Smad2 binds to Trim33 in the nucleus and also plays a role in MEE apoptosis.

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Figure 5-4. Schematic representation of medial edge epithelium (MEE) specific cis-regulatory elements.

Three cis-regulatory elements were identified; one in the intergenic region between the Ift43 and Tgfb3 genes, and two small, non-coding, evolutionarily conserved regions in intron 2 of the Ift43 gene.

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